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宫颈癌微环境的研究进展汪景灏;张蓉【期刊名称】《中国临床医学》【年(卷),期】2014(000)006【总页数】3页(P750-752)【作者】汪景灏;张蓉【作者单位】南方医科大学附属奉贤医院妇产科，上海 201499;南方医科大学附属奉贤医院妇产科，上海 201499【正文语种】中文【中图分类】R737.33宫颈癌发病率居全球妇科恶性肿瘤的第2位。
宫颈癌患者中有80%来自发展中国家。
我国是宫颈癌的高发国家，近20年来我国宫颈癌患者早期检出率增加，且患者趋于年轻化[1]。
肿瘤微环境是肿瘤周围的内环境，包括肿瘤周围的血管、免疫细胞、成纤维细胞、信号因子及细胞外基质。
一方面，肿瘤通过释放细胞外信号调控微环境；另一方面，肿瘤微环境也影响肿瘤细胞的生长，对肿瘤的增殖、侵袭、迁移、黏附及新生血管的形成具有重要作用[2]。
因此，探讨肿瘤微环境与宫颈癌间的关系显得尤为重要，可能为宫颈癌的早期诊断和治疗提供新的思路。
1 肿瘤微环境中的细胞1.1 成纤维细胞成纤维细胞主要负责细胞外基质的重构，同时还可通过旁分泌生长因子来调控细胞的增殖、存活和死亡。
在肿瘤形成过程中，成纤维细胞迁移至肿瘤性部位并增殖，产生大量的胶原蛋白，同时招募炎性细胞，进一步促进了组织的异常调节[3]。
Pietras等[4]发现，在HPV16型宫颈癌小鼠模型中，与肿瘤相关的成纤维细胞的聚集先于血管形成。
研究者[5]在应用雌激素的HPV16转基因小鼠宫颈癌模型中发现，肿瘤招募巨噬细胞后诱导并维持了肿瘤血管的生成。
来源于异常宫颈和宫颈癌的成纤维细胞不能表达炎性信号，这提示有其他细胞参与肿瘤炎性反应。
1.2 炎性细胞炎性细胞浸润在肿瘤的发展与侵袭中具有重要作用，这些炎性细胞包括中性粒细胞、嗜酸性粒细胞、巨噬细胞、肥大细胞等[3]。
1.2.1 中性粒细胞中性粒细胞可通过释放趋化因子和蛋白酶以及招募特异性和非特异性免疫效应细胞来改变微环境[6]。
研究[7]发现，中性粒细胞与宫颈癌患者的分期有关。
Development and re finement of a high-ef ficiency gene-targeting system for Aspergillus flavusPerng-Kuang Chang ⁎,Leslie L.Scharfenstein,Qijian Wei,Deepak BhatnagarSouthern Regional Research Center,Agricultural Research Service,U.S.Department of Agriculture,1100Robert E.Lee Boulevard,New Orleans,LA 70124,United Statesa b s t r a c ta r t i c l e i n f o Article history:Received 7January 2010Received in revised form 19February 2010Accepted 4March 2010Available online 16March 2010Keywords:Aspergillus flavus A flatoxinFunctional genomics pyrGGene targeting Conidial pigmentAn ef ficient gene-targeting system based on impairment of the nonhomologous end-joining pathway and the orotidine monophosphate decarboxylase gene (pyrG )in Aspergillus flavus was established.It was achieved by replacing the ku70gene with the Aspergillus oryzae pyrithiamine resistance (ptr )gene and by inserting the Aspergillus parasiticus cypA gene into the pyrG locus.The utility of this system was demonstrated by disruption of nine candidate genes for conidial pigment biosynthesis.The gene-targeting frequencies ranged from 80to 100%.Two linked genes on chromosome 4,wA and olgA ,were con firmed to be involved in pigment formation.In contrast to the parental strain which produced yellowish-green conidia,the knockout mutants produced white and olive-green conidia,respectively.The system was further re fined by restoring the pyrithiamine sensitivity and uracil auxotrophy in the A.flavus transformation recipient with an engineered pyrG marker.The improvement allowed gene manipulation using the reusable pyrG marker as shown by the restoration of laeA -mediated a flatoxin production in an A.flavus laeA -deleted mutant.Published by Elsevier B.V.1.IntroductionAspergillus flavus is a main producer of carcinogenic a flatoxins and is a pathogen of many agricultural commodities.It is also the second leading causative agent of invasive and non-invasive aspergillosis (Hedayati et al.,2007).A flatoxins,if ingested,pose a great risk to human and animal health,hence,their levels are stringently regulated (Guzman-de-Pena and Pena-Cabriales,2005;Otsuki et al.,2001).Of 77countries having regulations limiting mycotoxins,48have speci fic regulatory levels for total a flatoxins in foodstuffs and 21have regulations for a flatoxins in feedstuffs (FAO,1997).Signi ficant economic losses thus can result from a flatoxin contamination of food and feed.The estimated A.flavus genome is 37Mb and contains about 12,000genes (Payne et al.,2006).Genomic resources such as whole genome sequence and EST of A.flavus play an increasingly important role in the understanding of a flatoxin biosynthesis and fungus –plant interactions (Payne et al.,2006;Yu et al.,2004).Equally important is their impact on the understanding of A.flavus pathogenicity in humans and animals.The functions of the majority of A.flavus genes,however,are unknown.Although DNA microarrays allow genome-wide gene expression and association to be studied (Cary et al.,2007;Wilkinson et al.,2007),gene targeting along with subsequent genetic complementation to regain lost traits is still the best approach for understanding gene function.Historically,only Neurospora crassa orotidine monophosphate decarboxylase gene,pyr-4(Woloshuk et al.,1989),is frequently used for A.flavus transformation.A few others including A.flavus β-tubulin gene (Seip et al.,1990)and A.parasiticus nitrate reductase gene (Duran et al.,2007)are occasionally used.The ble gene for resistance to the antibiotic phleomycin has been demonstrated as a positive selectable marker for A.flavus (He et al.,2007);its ef ficacy is still under evaluation.Resistance markers developed from closely related A.oryzae ,such as the ptr pyrithiamine resistance gene (Kubodera et al.,2000)and a mutated succinate dehydrogenase gene,sdhB (cxr),resistant to carboxin,a systemic fungicide,developed for A.oryzae and A.parasiticus are likely applicable to A.flavus (Shima et al.,2009).High-throughput gene functional analysis requires an ef ficient system to reduce time and labor involved in identifying gene knockouts.Signi ficant increases in gene-targeting frequencies have been reported by disabling components of the nonhomologous end-joining (NHEJ)pathway,such as DNA-dependent protein kinase catalytic subunits of Ku70and Ku80,and DNA ligase IV (da Silva Ferreira et al.,2006;Meyer et al.,2007;Mizutani et al.,2008;Ninomiya et al.,2004;Takahashi et al.,2006).Adopting available protocols,demonstrating their ef ficacy and establishing a useful system for A.flavus remains a daunting task.This is particularly true when a system,in terms of selectable marker and recipient strain,is required for multiple rounds of gene-targeting experiments or for reintroducing targeted genes into a knockout strain.The inadequacy of current systems in these aspects has,in part,hampered the progress of the functional genomics in A.flavus .In this study,we first established a gene-targeting system for A.flavus by replacing ku70with A.oryzae ptr and by creating a uracilJournal of Microbiological Methods 81(2010)240–246⁎Corresponding author.Tel.:+15042864208;fax:+15042864419.E-mail address:perngkuang.chang@ (P.-K.Chang).0167-7012/$–see front matter.Published by Elsevier B.V.doi:10.1016/j.mimet.2010.03.010Contents lists available at ScienceDirectJournal of Microbiological Methodsj o u r na l h o m e p a g e :w w w.e l se v i e r.c o m /l o c a t e /j m i c m e t hauxotroph using a knock-in technique(Bardiya and Shiu,2007;Skory et al.,1990).We demonstrated its utility by disrupting several candidate genes and identified two genes involved in conidial pig-ment biosynthesis.We further refined this system by restoring pyrithiamine sensitivity with an engineered pyrG marker for subse-quent re-creation of the uracil auxotrophy.The incorporation of the reusable pyrG marker to the resulting recipient strain makes this improved system facilitate functional genomics study.2.Materials and methods2.1.Fungal strainsThe A.flavus CA14which produces aflatoxins and large sclerotia (Hua et al.,2007)was isolated from a pistachio bud in the Wolfskill Grant Experimental Farm(University of Davis,Winters,California, USA).CA14N1is a nitrate-nonutilizing strain derived from CA14.On a Difco™Czapek Solution Agar(CZ,Becton and Dickinson Company, Sparks,Maryland,USA)plate it exhibited expansive mycelial growth but barely produced conidia due to its inability to use nitrate,which is the sole nitrogen source of CZ.2.2.Construction of A.flavus ku70disruption vectorIn silico identification of the A.flavus ku70gene was performed initially on the5X draft database of the A.flavus genome sequence (http://www.aspergillusfl/genomics/).The ku70disruption vector was constructed in three steps.First,a0.5kb ku70coding region at the5′end(Fig.1A)was amplified by PCR using primers ku94H and ku600P(see Table1for all primers with designations and sequences).The PCR fragment after digestion with HindIII and PstI was cloned into the corresponding sites in pUC19.Second,a0.5kb coding region at the3′end was generated by PCR with ku1650P and ku2150K.The fragment was cloned into the vector obtained from the first step.Third,the2.0kb A.oryzae ptr marker amplified from pPTR1 (TaKarRa,Japan)with ptrU730P and ptr1230P was digested with PstI and cloned into the PstI site of the above construct.The disruption vector,pAfKuDV,was linearized by FastDigest®HindIII and KpnI in a universal buffer(Fermentas,Glen Burnie,Maryland,USA)prior to fungal transformation.Approximately5μg DNA was used in each of the two transformation experiments.2.3.Preparation of protoplasts and transformationIn the initial transformation for the disruption of the ku70gene, approximately108conidia harvested from V8agar plates(Chang and Hua,2007)were inoculated into100ml Czepak-Dox Broth(Becton and Dickinson)supplemented with0.5%casamino acids.The culture was shaken at150rpm for11–12h at30°C.Mycelia were collected on a 100μm nylon cell strainer,transferred to a50ml tube,and resuspended in 20ml offilter-sterilized enzyme solution that contained200mg lysing enzymes from Trichoderma harzianum(L1412,Sigma,St.Louis,Missouri, USA),50mg driselase,(Sigma),and40mg cell-wall digesting enzyme (Applied Plant Research,The Netherlands)in0.55M KC1,0.05M citric acid,pH5.8.The digestion was allowed to progress for2–3h at30°C with shaking(60rpm).Protoplasts were harvested byfiltering through a 40-μm nylon cell strainer and pelleted using a microcentrifuge.The protoplasts were washed twice with a solution of0.6M KC1,50mM CaC12and10mM Tris–HCl,pH8.0.Fungal transformation was performed as previously described with minor modifications(Horng et al.,1990). Polyethylene glycol(PEG)solution consisting of40%(w/v)PEG4000 (Fluka,Germany),0.6M KCl,50mM CaC12and10mM Tris–HC1,pH8.0 was used instead.The transformation mixture was added to CZ regeneration medium containing0.6M KCl,5mM ammonium sulfate, and0.1μg pyrithiamine(PT)/ml.Plates were incubated at30°C for up to 10days.2.4.Confirmation of gene disruption by PCRConidia of PT-resistant colonies were inoculated into1ml Potato Dextrose Broth(PDB;EMD Chemicals Inc.,Damstadt,Germany)in a 2-ml microfuge tube.The tube was incubated horizontally at30°C for 18to24h.Harvested mycelia were processed using a Scientific Industries'Disruptor Genie™(ZYMO RESEARCH,Orange County, California,USA).Genomic DNA was prepared using the ZR Fungal/ Bacterial DNA Kit™(ZYMO RESEARCH).Paired primers,based on specific locations in the expected genomic pattern generated after disruption of ku70by homologous recombination,were used in PCR (see Fig.1).They were set1,ku900and ku1500,set2,ku2210and ptr1,and set3,ku60and ptr800.PCR was carried out under the following conditions in a PERKIN ELMER GeneAmp PCR System2400. Fifty pmol of each primer and about10ng genomic DNA were added to50μl Platinum Blue PCR Supermix(Invitrogen,Carlsbad,California, USA)and subject to30cycles consisting of denaturation at94°C for 30s,annealing at55°C for30s and extension at72°C for2.0min. Similar PCR approaches were used to confirm other gene disruption events(seebelow).Fig.1.Replacement of A.flavus ku70by the ptr selectable marker.(A)Diagram depicting the replacement via double-crossover recombination.(B)PCR analyses of genomic DNA patterns of the recipient,R,and the ku70disruptant,D.The primers used were1:ku900 and ku1500,2:ptr1and ku2210,and3:ptr800and ku60.The DNA size markers(in kb) are lambda DNA/Hind III andØX174RF DNA/HaeIII fragments.241P.-K.Chang et al./Journal of Microbiological Methods81(2010)240–2462.5.Generation of a ku70-and pyrG-deleted strainA gene knock-in strategy(Bardiya and Shiu,2007)was adopted to generate a pyrG-deleted recipient rather than resorting to mutagen or UV treatment.Construction of the pyrG knock-in vector included cloning pyrG-associated fragments to theflanking regions of A. parasiticus cypA,a major part of which is missing in A.flavus(Ehrlich et al.,2004).A1.0kb5′-untranslated region(UTR)plus coding region and a1.0kb3′UTR plus downstream region of pyrG were generated by PCR using primers pg5HK and pg3Sp,and pg5Sp and pg3Sc,respec-tively.The primers cy280and cy550were used to amplify a2.7kb A. parasiticus cypA-containing fragment.These fragments containing tagged or native restriction sites were cloned sequentially into pUC19. The resulting vector was linearized by KpnI and SacI digestion prior to transformation.Preparation of protoplasts and fungal transformation were performed as described above except that,after transformation, PEG was removed by centrifugation.The protoplasts,after regeneration overnight at30°C in PDB containing0.6M KCl and uracil(2mg/ml), were spread onto PDA plates which contained0.6M KCl,uracil(2mg/ ml),and5-fluoroorotic acid(FOA,2mg/ml).Disruption of pyrG in a transformant was confirmed by PCR with the primers pg5HK and pg3Sc. The generatedΔpyrG strain lacked230nt in the pyrG3′coding region plus60nt in3′UTR.e of pyrG-based disruption vectors for identifying genes involved in conidial pigment biosynthesisOnly a few Aspergillus genes have been reported to be involved in conidial pigment biosynthesis,i.e.A.fumigatus alb1,abr1,abr2(a laccase gene=A.nidulans yA)and a yg1(Tsai et al.,1999)and A.nidulans wA (=A.fumigatus alb1)(Mayorga and Timberlake,1992).Homologues of the above genes and several genes encoding laccases that may be involved in pigment formation were identified by BLAST(Schaffer et al., 2001)search of the Aspergillus Comparative Database at Broad Institute (/annotation/genome/aspergil-lus_group/MultiHome.html).Restriction analyses on the identified genes and theirflanking regions were carried out using the DNAMAN software(Lynnon Soft,Vandreuil,Quebec,Canada).DNA fragments specific to theflanking regions of a gene to be targeted were amplified by PCR,digested with appropriate restriction enzymes(Fementas),and inserted into unique sites of pPG28,which contains the A.parasiticus pyrG gene in a 2.7-kb BamHI–SalI fragment(GenBank accession number:EU879956,Fig.2A).The PCR primers used are listed in Table2.Mycelia for the preparation of protoplasts were obtained from conidia of theΔpyrG mutant grown in PDB containing0.5mg uracil/ml shaken at150rpm for20h at30°C.The disruption vectors were linearized prior to transformation to yield DNA ends that are identical to the targeting sites.2.7.Replacement of the inserted ptr marker by a reusable pyrG selectable markerA gene replacement protocol was used to restore the pyrithiamine sensitivity of thefirst createdΔpyrG transformation recipient strain.A 1.4kb5′-UTR of ku70was amplified by PCR using primers ku5Sm and ku5X.A1.7kb3′-UTR of ku70was amplified using primers ku3X and ku3S.The two fragments were cloned sequentially into the corresponding sites in pUC19to give pKu5+3.A direct repeat-recombination strategy was the basis for generating a reusable pyrG marker used to replace the ptr dominant selectable marker.To this end,the R region in Fig.2A was amplified with primers pgXho and pgSal.The PCR fragment after digested by XhoI and SalI was used to replace the1.0kb XhoI–SalI region(right side of Fig.2A),which resulted in a pyrG markerflanked by two0.5kb direct repeats(R-pyr-R);a putative polyadenylation signal,AATAA,is located about10nt before the XhoI site.The R-pyr-R fragment generated after BamHI and SalI digestion was cloned into the XbaI site of pKu5+3by blunt-end ligation.The resulting vector was digested with HindIII and SmaI(Fementas)prior to transformation.Table1Oligoprimer designations and their sequences.Primer Sequenceku94H CTGAAAGCTTCGGAGGCTAACku600P ATACTGCAGGGTCATCATTATCGGTGACTku1650P CTTCCTGCAGAGAAACTCTGGku2150K TGAGGAACAGGTACCGTAAGC.ku60CGACGAGAGTGTACACACTTku270CGGATGAGTTGGAGCTGAAGku900TCAAGATCTGTCCCACGGku1500TGACGGACGTCATCAGCGku2210TCCACACGCTCAACGAGATCku2540GACTGCAACGTTGCTGGTACku5Sm CTTCGCCCGGGTACGGGTCACCTAATCku5X TATCTAGAGTCGTAAGTCATGAATTGCGTku3X ATTCTAGACAACGCTAGTATTGGTTACGAGku3S CTAGAACGAATTCGTGTCGACACTGAptrU730P ATACTGCAGACGGGCAATTGATTACGptr1230P TTACTGCAGCCGCTCTTGCATCTTTGptr1TGGCAGCTGGAGGAGACATGptr800CCTTCTGTGCGAAGCGCTTGpg5HK ATTAAGCTTATTGCTATGTCCCTGAAAGpg3Sp ATTGCATGCTAACTTCAGACTGAACCTCpg5Sp TATGCATGCACTCGAAATGACTACTACTATpg3Sc TGAGTCTAGCTGAGCTCGGCTCpgXho ATACTCGAGATCTCAGAACAATATACCAGpgSal ATAGTCGACCGGCTTATTCAGTAGATTcy280AATGCTAGCTTGTGTGGATTCGTGAGTGTCcy550ATAGCTAGCATTGCTCTGCATACTCGGAClaeA5Sp CGATTAGTTCGTTGAACTGTCAlaeA5S AATGTCGACTGTGAGTAGTACGAGTCGlaeA3B ATAGGATCCACA AATTATTCACGGTGlaeA3Sc TGGCACCACACAAGCTCATATClaeA243ATAGTCGACTTACCGGACAGTGCAAGGlaeA2897TAAGTCGACAAGAGCTGCATCGCGATGTAtfR5Sp CCGGCATGCCTAGACAGACAATCACtfR5S TATGTCGACCTCACGTCTGTGCAGGCCtfR3B GTCGGATCCACATCAAAGAGGGATACTtfR3SmATAGCCCGGGTAATGTCGTTGGTCFig.2.The original and the reusable pyrG markers for fungal transformation.(A)The2.7kb BamHI–SalI fragment that contains the A.parasiticus pyrG gene inserted into themultiple-cloning-site region of pUC19(pPG28).E,EcoRI*;Sc,SacI*;K,KpnI;Sm,SmaI*;B,BamHI*;P,PstI;Xh,XhoI*;S,SalI*;Sp,SphI*;H,HindIII*.The symbol*indicatesunique restriction sites.(B)Schematic representation of the forced recombinationbetween the R repeat regions under FOA positive selection to regain uracil auxotrophy. 242P.-K.Chang et al./Journal of Microbiological Methods81(2010)240–2462.8.Determination of frequencies of self-resolution of the reusable pyrG marker on different mediaConidia were harvested from a PT-sensitive strain,whose previously inserted ptr marker had been replaced by the reusable pyrG (R-pyr -R)marker.Different culture media were used to examine the selection ef ficiency for the resolved auxotrophic mutants.Approximately 105,106,and 107conidia were spread onto PDA plates which contained 2mg uracil/ml and 2mg FOA/ml,and CZ(NH 4+)plates which contained 2mg uracil/ml,2mg FOA/ml,and 5mM ammonium sulfate.The plates were incubated at 30°C for 7to 10days.The ku70-speci fic primers ku270and ku2540were used to con firm forced resolution (loop-out)of the pyrG marker leaving only one copy of the R region in the ku70locus (Fig.2B).2.9.Deletion of laeA by R-pyrG-R and re-creation of a uracil auxotrophic ΔlaeA mutantUsing similar steps described in Section 2.2,we constructed a laeA -disruption vector based on the R-pyr -R marker with the following primers:laeA5Sp,laeA5S,laeA3B,and laeA3Sc to amplify the two flanking regions used in targeting laeA via double-crossover recombi-nation.The resulting vector was linearized with SphI and SacI prior to transformation.Putative ΔlaeA mutants were selected based on changes in colony morphology and loss of a flatoxin production.Disruption of laeA was con firmed by PCR analyses of the genomic patterns of the transformants (data not shown).Approximately 2×106conidia of a con firmed ΔlaeA mutant were spread onto CZ(NH 4+)plates,which contained 2mg uracil/ml,2mg FOA/ml,and 5mM ammonium sulfate,for the generation of uracil auxotrophic ΔlaeA mutants.2.10.Reintroduction of full-length genomic laeA into an A.flavus ΔlaeA mutantThe laeA gene was reintroduced into the ctfR2gene locus (AFL2G_07245.2)located in a subtelomeric region of chromosome 3.Disruption of ctfR2had no effect on the production of a flatoxin,cyclopiazonic acid,nor on gross morphology (unpublished results).The primer pair,tfR5Sp and tfR5S,and the primer pair,tfR3B and tfR3Sm were used to amply a 5′and a 3′regions of ctfR2,respectively.The two PCR fragments were cloned into corresponding sites in pUC19followed by insertion of the A.parasiticus pyrG gene.The full-length laeA gene was ampli fied from A.flavus CA14genomic DNA template using Accu-Prime ™Pfx PCR Supermix (Invitrogen)with primers laeA243and laeA2897each tagged with a SalI site.The 2.7kb laeA -containing SalI –SalI fragment was cloning into the SalI –XhoI sites after the 1.2kb SalI –XhoI fragment downstream of the pyrG gene (see Fig.2A)was removed.The resulting targeting vector was digested with HindIII and SphI prior to transformation.The primers laeA243and laeA2897were used in PCR to con firm the presence of the full-length laeA gene in the comple-mented transformants.2.11.Thin-layer chromatography (TLC)analysis of a flatoxin B 1The transformants on the regeneration plates (PDA supplemented with 0.6M KCl)putatively complemented by the full-length laeA were transferred onto PDA plates for TLC analysis.One PDA agar plug was cored from a 5day-old transformant culture plate grown at 30°C,placed into a microfuge tube and extracted with 0.2ml of acetone for 1h.Ten microliter of each extracts was spotted onto a Si250TLC silica gel plate (J.T.BAKER,Phillipsburg,New Jersey,USA).The metabolites were resolved with a solvent system of toluene:ethyl acetate:acetic acid (80:10:10,vol/vol/vol).3.Results3.1.Replacement of ku70by the dominant ptr selectable marker Growth of A.flavus CA14and CA14N1was inhibited at 0.01μg PT/ml.Therefore,0.1μg PT/ml was chosen as the selection concentration.Two independent transformation experiments yielded a total of 102PT-resistance colonies.PCR analyses of the genomic DNA from 80transformants showed that two transformants were ku70knockouts.Deletion of ku70was con firmed with primers speci fic to the ku70coding region,ptr downstream and upstream regions,and regions beyond the expected homologous recombination sites (Fig.1A).No PCR products were generated from the genomic DNA of the ku70knockout when primers ku900and ku1500were used.In contrast,these primers yielded a 0.6kb PCR fragment from the recipient's genomic DNA (Fig.1B).As expected,primers ptr1and ku2210,which amplify a small region beyond the expected integration site i.e.,ku2150and the upstream region of ptr (Fig.1A),generated a 1.4kb PCR fragment from the genomic DNA of the ku70knockout.Likewise,primers ptr800and ku60yielded a 1.0kb PCR fragment,expected from another flanking region after ku70was targeted by ptr through homologous integration.The two regions were not present in the recipient's genome.These results showed that the ptr marker had replaced the ku70gene in the A.flavus genome.e of the Δku70ΔpyrG strain to identify conidial pigment biosynthetic genesThe ku70-defective genetic background and the positive FOA selection facilitated the generation of ΔpyrG mutants that produced white-colored conidia on regeneration plates (data not shown).A Δku70ΔpyrG strain was used as the transformation recipient in the identi fication of conidial pigment genes.Of the nine genes disrupted two genes,AFL2G_09923.2and AFL2G_09924.2,were con firmed to be involved in conidial pigment biosynthesis (Table 2).The AFL2G_09923.2knockouts produced expected white conidia while knockouts of AFL2G_09924.2,a homolog of A.fumigatus abr2(br:brown)and A.nidulans yA (y:yellow),produced olive-green conidia not reported before in Aspergillus species (Fig.3A).They were named wA and olgA ,Table 2PCR primers used in the construction of disruption vectors for identifying conidial pigment genes.Gene locusa5′Flanking region3′Flanking regionGeneAFL2G_00330.2cacggcccgggatgtcacta gtatcgggatccaccttccctcctt cggtttgagcaagcttagaatc gctggaagatcgtcgaccctgtgaa ayg1AFL2G_09923.2gcagcttataggagaattcact gccgactggatccctctgtgtattgtcgacttccgatactatcta caatgcatgcaatacttaccgatg alb1(wA )AFL2G_09924.2acgtattggtagcatgctcacgc tgcgtcgacagctttaagccacgcggc ttcgaagagctccggtctttccgt catggatccacgagcacccgatgt abr2(yA )A.flavus olgA AFL2G_09962.2atggaacatcacccgggtattggcca tatggatccaatgatgagatgtcag tacgtcgacttcaaacttgtccctg tcacttgcgtcgcatgcctg abr1AFL2G_08008.2cctatctggtgcatgcggtgaacttg gttgcattgagtcgacgccaggc gatgaggaggatccctacattg gtcgagtacagaattcgaact lac1AFL2G_09420.2cgcgtttaattcggcatgca ggaagtcgacacgggcagtg ctagtggatccatcccacaga cgagattggaattccccggg lac2AFL2G_10583.2ttgctgggagcatgcctgtatcac gttcatggtcgacaaccatac cgaacagcggatccactgtattg catgcttggcttgtttgatcglac3AFL2G_11132.2ctacggcatgcccgggtgacacag atcgcgtcgacacggatagg tataggatccgcgctgccaac atgggaattcccgggataagacatgg lac4AFL2G_11750.2cgtgcgtcactatgcatgcagg catcatgggtctgtcgacgccatcggtgatggtgacacagctggtcaa cacaagaactagaattcaagacgglac5a/annotation/genome/aspergillus_group/MultiHome.html .243P.-K.Chang et al./Journal of Microbiological Methods 81(2010)240–246respectively.The con firmed knockouts of each of the rest seven genes including homologs of A.fumigatus abr1and ayg1produced yellowish-green conidia same as those of the A.flavus parental strain (data not shown).The overall gene-targeting frequencies estimated from all the experiments ranged from 80%to 100%.The locations of these genes were tentatively assigned to the A.flavus genome using the chromo-somal map of A.oryzae (http://www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao ).The wA and olgA genes are clustered on chromosome 4,and AFLG_09962.2,a homolog of A.fumigatus abr1(Tsai et al.,1999),is located about 100kb away.Fig.3B shows the genomic patterns of some of the knockouts compared to those of the recipient strain as con firmed by PCR with primers encompassing the targeting region.They were 4.3kb vs.1.7kb for AFL2G_09962.2,4.1kb vs.1.5kb for olgA ,4.7kb vs.3.5kb for wA ,and 5.0kb vs.2.3kb for AFL2G_00330.2.3.3.Restoration of PT sensitivity and uracil auxotrophy in the original recipientA drawback of many developed transformation systems based on single selectable markers is the loss of auxotrophy or drug sensitivity after transformation.In this study,the ptr marker was readily replaced by the reusable pyrG marker (R-pyr -R)which contains two direct repeats of a 5′UTR region (Fig.2B),and the uracil auxotrophy wasrecovered from FOA selection.The estimated frequencies of self-resolution of the R-pyr -R marker reached 100%on CZ(NH 4+)and PDA media.The PTs Δku70ΔpyrG derivatives containing a single copy of the 5′UTR (R)were easily generated on both media (Fig.4A and B.Although CZ(NH 4+)yielded smaller colonies than PDA,it gave more than 5-fold higher ΔpyrG derivatives than PDA (Table 3).3.4.Demonstration of the ef ficacy of the improved system by reintroducing laeATo test the utility of the developed system,we disrupted the laeA gene,a major regulatory gene of secondary metabolism (Bok and Keller,2004),in a PTs Δku70ΔpyrG strain (data not shown),regenerated the uracil auxotrophy by forcing out the reusable pyrG marker,and restored the lost a flatoxin-producing ability by reintro-ducing an intact copy of laeA via a new round of transformation based on the pyrG selectable marker.Five of the six transformants examined produced a flatoxin B 1(Fig.5A),and the result was consistent with the presence of the intact laeA gene in the a flatoxin-producing transfor-mants (Fig.5B).4.DiscussionWe combined available selectable markers for fungal transforma-tion and formulated an ef ficient gene-targeting protocol for A.flavus .The demonstrated ef ficacy of this system indicates that the heterol-ogous genes are functional in A.flavus .An important consideration for high-throughput functional genomics is to minimize the time in selecting correct gene knockouts,that is,only a very small number of transformants should be examined before one is identi fied.The ku70gene is critical for DNA repair via the nonhomologous end-joining (NHEJ)pathway,and its impairment greatly reduces the frequency of heterologous integration of transforming DNA.The gene-targeting frequencies of the NHEJ-de ficient A.flavus system are well withintheFig.3.Identi fication of conidial pigment genes.(A)Colony morphology of AFL2G_09962.2(top),AFL2G_09924.2(olgA ,bottom left),and AFL2G_09923.2(wA ,bottom right)disruptants after growth at 30°C for 5days on PDA.(B)Con firmation of disruption of AFL2G_09962.2(9962),oligA ,wA and AFL2G_00330.2(0330).M:1kb DNA ladder,A:recipient strain,B:disruptant.Fig. 4.Determination of self-resolution frequencies on different media.(A)Colony morphology of FOA-resistant mutants.(B)PCR con firmation of deletion of the pyrG marker.C:PTs Δku70;1–4:independent derivatives.The lack of products in C was due to the presence of the R repeats in the R-pyrG -R marker which interfered PCR ampli fication.244P.-K.Chang et al./Journal of Microbiological Methods 81(2010)240–246ranges of what have been reported for a single homologous insertion in other fungi(da Silva Ferreira et al.,2006;Ninomiya et al.,2004; Takahashi et al.,2006).The generation of the A.flavus uracil auxotrophic mutant by the knock-in strategy based on FOA resistance can be easily adopted for other fungi.This approach eliminates the common practice of mutagen treatments which often result in genetic changes.Although these changes are not always morphologically apparent,they may complicate functional studies.A drawback of past developed transformation systems for A.flavus and many other fungi is the lack of double selectable markers and double mutants suitable for gene manipulation,for example gene knockout experiments followed by genetic complementation.The creation of necessary mutants by conventional approaches is a formidable task.The experimental steps can be adopted and used to readily generate a highly efficient system that consists of a reusable selection and second selection as the described uracil auxotrophy and pyrithiamine sensitivity to meet the prerequisites.It can be achieved byfirst knocking out an NHEJ-associated gene with either an auxotrophic marker,such as the nitrate reductase gene(niaD)in a spontaneous niaD mutant(Malardier et al., 1989;Pereira et al.,2004;Whitehead et al.,1990)or with a dominant marker,such as the aforementioned ptr,ble and sdhB(cxr)genes(He et al.,2007;Shima et al.,2009)to facilitate subsequent rounds of gene-targeting.The refined system has been used successfully to disrupt and reintroduce a gene,such as laeA,a regulatory gene of secondary metabolism(Bok and Keller,2004)into a specific genome locus to restore the lost aflatoxin production(Fig.5).The NHEJ-deficient background of the recipient stain also allows integration of a circular vector containing sequence regions identical to a genomic portion via singe-crossover recombination.Based on pyrG we used this approach and genetically complemented an A.flavus knockout mutant of msnA (unpublished results),the orthologue of Saccharomyces cerevisiae MSN2necessary for cells to cope with a broad range of stresses(Ruis and Schuller,1995).The added benefit of this system includes the restored pyrithiamine sensitivity,upon which the dominant ptr marker can be used as an alternative in double knockout or genetic complementation experiments to provide greater versatility. AcknowledgmentWe thank Alice Yeh of University of Virginia for technical assistance.ReferencesBardiya,N.,Shiu,P.K.,2007.Cyclosporin A-resistance based gene placement system for Neurospora crassa.Fungal Genet.Biol.44,307–314.Bok,J.W.,Keller,N.P.,eA,a regulator of secondary metabolism in Aspergillus spp.Eukaryot.Cell3,527–535.Cary,J.W.,O'Brian,G.R.,Nielsen,D.M.,Nierman,W.,Harris-Coward,P.,Yu,J.,Bhatnagar,D.,Cleveland,T.E.,Payne,G.A.,Calvo,A.M.,2007.Elucidation of veA-dependentgenes associated with aflatoxin and sclerotial production in Aspergillusflavus by functional genomics.Appl.Microbiol.Biotechnol.76,1107–1118.Chang,P.-K.,Hua,S.S.,2007.Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillusflavus.Lett.Appl.Microbiol.44,131–137.da Silva Ferreira,M.E.,Kress,M.R.,Savoldi,M.,Goldman,M.H.,Hartl,A.,Heinekamp,T., Brakhage, A.A.,Goldman,G.H.,2006.The akuB(KU80)mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus.Eukaryot.Cell5,207–211.Duran,R.M.,Cary,J.W.,Calvo,A.M.,2007.Production of cyclopiazonic acid,aflatrem, and aflatoxin by Aspergillusflavus is regulated by veA,a gene necessary for sclerotial formation.Appl.Microbiol.Biotechnol.73,1158–1168.Ehrlich,K.C.,Chang,P.-K.,Yu,J.,Cotty,P.J.,2004.Aflatoxin biosynthesis cluster gene cypA is required for G aflatoxin formation.Appl.Environ.Microbiol.70,6518–6524. 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Kubodera,T.,Yamashita,N.,Nishimura,A.,2000.Pyrithiamine resistance gene(ptrA)of Aspergillus oryzae:cloning,characterization and application as a dominant selectable marker for transformation.Biosci.Biotechnol.Biochem.64,1416–1421. Malardier,L.,Daboussi,M.J.,Julien,J.,Roussel,F.,Scazzocchio,C.,Brygoo,Y.,1989.Cloning of the nitrate reductase gene(niaD)of Aspergillus nidulans and its use for transformation of Fusarium oxysporum.Gene78,147–156.Mayorga,M.E.,Timberlake,W.E.,1992.The developmentally regulated Aspergillus nidulans wA gene encodes a polypeptide homologous to polyketide and fatty acid synthases.Mol.Gen.Genet.235,205–212.Meyer,V.,Arentshorst,M.,El-Ghezal,A.,Drews,A.C.,Kooistra,R.,van den Hondel,C.A., Ram,A.F.,2007.Highly efficient gene targeting in the Aspergillus niger kusA mutant.J.Biotechnol.128,770–775.Mizutani,O.,Kudo,Y.,Saito,A.,Matsuura,T.,Inoue,H.,Abe,K.,Gomi,K.,2008.A defect of LigD(human Lig4homolog)for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae.Fungal Genet.Biol.45,878–889. Ninomiya,Y.,Suzuki,K.,Ishii,C.,Inoue,H.,2004.Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining.Proc.Natl.Acad.Sci.USA101,12248–12253.Otsuki,T.,Wilson,J.S.,Sewadeh,M.,2001.What price precaution?European harmoniza-tion of aflatoxin regulations and African groundnuts exports.Eur.Rev.Agric.Econ.28, 263–284.Payne,G.A.,Nierman,W.C.,Wortman,J.R.,Pritchard, B.L.,Brown, D.,Dean,R.A., Bhatnagar,D.,Cleveland,T.E.,Machida,M.,Yu,J.,2006.Whole genome comparison of Aspergillusflavus and A.oryzae.Med.Mycol.44(Suppl),9–11.Table3Self-resolution frequencies of the R-pyrG-R selectable marker on media supplemented with FOA.Medium Number of spores FOA-resistant colony a Frequency(%)bCZ(NH4+)1051±1–c10612±3100107113±11100PDA1050±0–1061±1–10718±6100a Average±SD.b Frequency was estimated based on four independent colonies examined.c Notdetermined.Fig. plementation of aΔlaeA strain with an amplified genomic laeA fragment.(A)Restoration of aflatoxin in the transformants(B)Confirmation of the presence of the introduced laeA in the aflatoxin-producing transformants.M:DNA1-kb ladder.Transfor-mants1to6are the same in A and B.245P.-K.Chang et al./Journal of Microbiological Methods81(2010)240–246。
重磅解读RNA编辑系统CRISPR-Cas13a发展脉络CRISPR/Cas系统是目前发现存在于大多数细菌与所有的古菌中的一种免疫系统，被用来识别和摧毁抗噬菌体和其他病原体入侵的防御系统。
在CRISPR/Cas系统中，CRISPR是规律间隔性成簇短回文重复序列（clustered regularly interspaced short palindromic repeats）的简称，涉及细菌基因组中的独特DNA区域，也是储存病毒DNA片段从而允许细胞能够识别任何试图再次感染它的病毒的地方，CRISPR 经转录产生的RNA序列（被称作crRNA）识别入侵性病毒的遗传物质。
Cas是CRISPR相关蛋白（CRISPR-associated proteins, Cas）的简称，Cas蛋白像一把分子剪刀那样切割细菌基因组上的靶DNA。
CRISPR/Cas系统是古菌和细菌的抵抗病毒和质粒侵染的重要免疫防御系统。
CRISPR-Cas系统划分为两大类，第一大类CRISPR-Cas系统由多亚基组成的效应复合物发挥功能；第二大类是由单个效应蛋白（如Cas9, Cpf1, C2c1、C2c2 等）来发挥功能。
其中，Cas9, Cpf1, C2c1和C2c2（也被称作Cas13a）均具有RNA介导的DNA核酸内切酶活性。
目前，Cas9和Cpf1蛋白作为基因组编辑工具被广泛应用，克服了传统基因编辑技术步骤繁琐、耗时长、效率低等缺点，以其较少的成分、便捷的操作以及较高的效率满足了大多数领域的基因编辑需求，并有着潜在且巨大的临床应用价值。
然而，目前得到广泛应用的CRISPR/Cas9系统经常会发生脱靶效应，导致科学家们不想要的结果。
而新发现的CRISPR-Cas13a似乎具有更强的特异性，或许有助克服这一点。
为了让读者更好地了解CRISPR-Cas13a系统的研究脉络，谷君在/pubmed网站上以CRISPR和（Cas13a or Cas13 or C2c2）为关键词，除去不相关的检索结果和综述论文类型的检索结果，获得11项相关的检索结果。
《原发性开角型青光眼的遗传学研究》篇一一、引言原发性开角型青光眼（POAG）是一种常见的致盲性眼病，其特点是眼内压升高，但前房角保持开放。
该病具有显著的遗传倾向，家族聚集性明显。
近年来，随着遗传学技术的快速发展，对POAG的遗传学研究取得了重要进展。
本文旨在综述POAG的遗传学研究现状，探讨其遗传机制及未来研究方向。
二、POAG的遗传学背景POAG的遗传学研究主要关注其遗传模式、基因变异及遗传风险评估等方面。
根据研究，POAG的发病与多个基因的变异有关，具有多基因遗传特征。
此外，环境因素、生活方式等也会影响POAG的发病风险。
三、POAG的遗传学研究方法目前，对POAG的遗传学研究主要采用以下方法：1. 家族研究：通过收集POAG患者家族资料，分析家族内发病情况，探讨遗传因素在发病中的作用。
2. 基因组关联研究（GWAS）：利用大规模基因组关联分析，寻找与POAG发病相关的基因变异。
3. 候选基因研究：针对已知或假设与POAG发病相关的基因进行深入研究，分析其变异与疾病的关系。
4. 基因表达研究：通过分析基因表达谱，探讨POAG发病过程中基因表达的变化。
四、POAG的遗传学研究进展1. 家族研究：通过对POAG患者家族的调查，发现家族内发病率明显高于一般人群，表明遗传因素在POAG发病中起重要作用。
2. 基因组关联研究：GWAS已成为POAG遗传学研究的重要手段。
多项GWAS研究发现，多个基因变异与POAG发病相关。
然而，由于POAG具有复杂的多基因遗传特征，仍需进一步深入研究。
3. 候选基因研究：针对已知或假设与POAG发病相关的基因进行研究，如MYOC、OPTN等。
这些基因的变异可能影响眼内压调节、视网膜神经传导等过程，从而增加POAG的发病风险。
4. 基因表达研究：通过分析POAG患者与健康人群的基因表达谱，发现某些基因在POAG发病过程中表达异常。
这些基因可能参与眼内压调节、视网膜神经保护等过程，为POAG的发病机制提供新的线索。
兽医临床科学 | Veterinary clinical science1212020.19·0 引言临床上引起仔猪出现腹泻疾病的原因十分复杂，通常与病原微生物侵入及不合理的养殖管理模式有很大联系。
仔猪腹泻疾病的诊断可结合流行病学、临床症状、病理变化作出初步诊断，但是腹泻疾病造成的很多病理变化和临床症状大致相同，如果兽医人员缺乏诊治经验，常会做出错误的判断，造成整体的治疗方案缺乏针对性。
因此面对复杂的仔猪腹泻致病原因，需要进行认真细致的分析，做好腹泻疾病的有效鉴别。
而仔猪腹泻发生后，在短时间内临床症状会加重，如果不能及时采取针对性措施进行治疗，造成严重死亡。
因此，在对腹泻疾病的患病猪进行治疗中可选择使用腹腔给药方式，保证药物直达病灶，提高治疗效果，缩短发病周期，提高治愈率。
1 发病经过2019年1月26日，某养殖户养殖的新生仔猪群中突然出现一种传染性疾病，该病发病速度较快，以整窝哺乳仔猪发病为主。
发病猪主要表现为厌食，体温升高，嗜睡，气喘，皮肤发紫，出现明显的腹泻症状，先是排出黄白色的粥样腹泻物，随后排出水样腹泻物。
出现临床症状1～2 d 后，患病猪机体严重脱水，最终衰竭而死。
该养殖场的整体防寒保暖性能较差，圈舍低温潮湿，粪便堆积严重，有害气体积累。
也没有对猪群进行妥善的疫苗免疫接种，卫生环境普遍较差。
2 临床症状养殖场新生仔猪群中突然出现个别发病猪，临床上表现为呕吐腹泻，粪便呈现青黄色，略带白色并且恶臭难闻。
出现临床症状后，养殖户立即将患病猪单独隔离，并使用青霉素链霉素进行治疗，效果不明显。
病情快速传播，蔓延整窝新生仔猪。
主要表现为黄色或黄白色的水样腹泻，在腹泻物当中还含有大量的凝乳块，患病时间较长的猪表现为精神萎靡不振，身体严重脱水并且很快死亡，发病急，发病过程短，造成的死亡率高[1]。
3 病理学变化通过对养殖场的猪进行解剖，发现大多数病死猪的仔猪腹泻鉴别和腹腔注射药物治疗乔启波（吉林省延边朝鲜族自治州动物疫病预防控制中心，延边 133000）摘要：仔猪腹泻疾病一直是困扰生猪养殖业的一大难题，尽管很多科研人员在疾病防治、营养管理和遗传学等方面取得突出成就，但是养殖场仔猪腹泻的发生率仍然较高，依然是养殖场的常发病和高发病。
Abstract Submittedfor the MAR15Meeting ofThe American Physical SocietyCarrier Mediated Ferromagnetism in Fe-doped SrTiO31CHUN-LAN MA,School of Mathematics and Physics,Suzhou University of Science andTechnology,Suzhou215009,China,ROCIO CONTRERAS-GUERRERO,RAVIDROOPAD,Ingram School of Engineering,Texas State University,San Marcos,TX78666,USA,BYOUNGHAK LEE,Department of Physics,Texas State Uni-versity,San Marcos,TX,78666,USA—The discovery of III-V dilute magneticsemiconductors(DMSs)and the subsequent unsuccessful search for room temper-ature ferromagnetism in DMSs have motivated researches on alternate dilute mag-netic systems.Recent progresses in thinflim growth techniques of perovskite oxidessuggest that dilute magnetic oxides(DMOs)can be viable candidates to improvethe magnetic properties of DMSs.In this talk we present an ab initio study of Fe-doped SrTiO3.Wefind that a ferromagnetic ordering among localized Fe t2g spinsis mediated by itinerant Fe e g electrons.The exchange interaction between t2g ande g electrons depends on crystalfield splitting,on-site electron-electron interaction,and the relative energy of Fe d-ortbitals to oxygen p-orbitals.The exchange couplingand the majority-minority spin splitting decrease with decreasing carrier concentra-tion,confirming that itinerant carriers mediate the ferromagnetism.1C.Ma is supported by NSF of China(Grant Nos.11247023and11304218),JiangsuQing Lan Project,and Jiangsu Overseas Research&Training Program.R.C.-G,R.D.,and B.L.are supported by AFOSR,award number FA9550-10-1-0133.Chun-Lan MaSchool of Mathematics and Physics,Suzhou University of Science and Technology,Suzhou215009,China Date submitted:15Nov2014Electronic form version1.4。
J Apoplexy and Nervous Diseases, July 2024, Vol 41，No. 7偏头痛发病机制及生物标志物研究进展毛西京， 朱博驰综述， 于挺敏审校摘要： 偏头痛是一种具有多种亚表型的异质性疾病，其诊断主要基于临床标准，缺乏特异性的生物标志物进行客观评估，影响了偏头痛的精确诊断、治疗选择以及预后评估。
近年来偏头痛在遗传、生化、影像等方面研究取得重大进展，为临床诊断及治疗偏头痛提供了客观的检测指标。
如能在临床工作中选择特异性、敏感性、易检测、可行性高的标志物将推动偏头痛早期诊断、精准化治疗的步伐。
关键词： 偏头痛； 生物标志物； 神经元； 胶质细胞中图分类号：R747.2 文献标识码：A Research advances in the pathogenesis and biomarkers of migraine MAO Xijing ，ZHU Bochi ，YU Tingmin. （The Sec⁃ond Hospital of Jilin University ， Changchun 130000， China ）Abstract ： Migraine is a heterogeneous disease with various subtypes ， and the diagnosis of migraine mainly relies on clinical criteria. The lack of specific biomarkers for objective assessment impacts the precise diagnosis ， treatment selec⁃tion ， and prognostic assessment of migraine. In recent years ， great progress has been made in migraine in terms of genet⁃ics ， biochemistry ，and imaging ， which provides objective indicators for the clinical diagnosis and treatment of migraine. Identifying specific ，sensitive ，easily detectable ，and highly feasible markers in clinical practice will accelerate the early di⁃agnosis and precise treatment of migraine.Key words ： Migraine ； Biomarkers ； Neurons ； Glial cells偏头痛的发病机制尚不完全明确，越来越多的研究发现神经元-神经胶质细胞-血管交互作用的炎性病理生理过程参与其中，并且从血液、脑脊液、唾液、影像检查中均发现了有意义的标志物，这些标志物成为偏头痛诊疗的潜在靶点。
补体缺陷及其疾病李圣杰;张爱平(综述);曹文俊(审校)【摘要】Complement system,which has the characteristics of crucial immune responses and cascade reactions, not only has a crucial role in acting as a bridge between innate and adaptive immunities,but also is a part of immune regulatory network,and has important significance in the preservation of immunological homeostasis.Early studies introduced complement component deficiency.The abnormal quality and quantity of complement suggest that complement deficiency is associated with an increasing prevalence of infections and autoimmune diseases.This paper reviews the current common complement deficiencies and their associated diseases,expecting to provide new thoughts for the laboratory diagnosis of diseases.%补体系统作为机体重要的免疫效应及其链式放大系统，是连接固有免疫和适应性免疫的重要桥梁，同时作为机体免疫调控网络的重要环节，在维持机体免疫自稳方面发挥重要作用。
中西医结合护理Chinese Journal of Integrative Nursing2023 年第 9 卷第 11 期Vol.9， No.11， 2023指压天突穴引咳法在早期肺癌术后患者中的应用边冠军1，2， 张春梅3， 刘丽峰2（1. 天津中医药大学， 天津， 301617；2. 天津医科大学肿瘤医院 国家肿瘤临床医学研究中心 天津市肿瘤防治重点实验室天津市恶性肿瘤临床医学研究中心 肺部肿瘤科， 天津， 300060；3. 天津中医药大学护理学院， 天津， 301617）摘要： 目的 探讨指压天突穴引咳法在早期肺癌术后患者中的应用。
方法 便利抽样法选取天津市某三级甲等肿瘤专科医院肺部肿瘤科2023年1月—3月收治的100例呼气峰值流速（PEF ）≤5.33L/s 的早期肺癌切除术的患者，随机分为观察组和对照组，各50例。
对照组接受常规护理措施，观察组在对照组基础上实施指压天突穴引咳法干预。
比较两组患者术后第1天雾化后咳出第一口痰的用时、术后3 d 晨起PEF 与入院PEF 差值、肺部并发症发生情况和咳痰效果。
结果 观察组咳痰效果评价优于对照组，差异有统计学意义（P ＜0.01）。
观察组术后第1天雾化后咳出第一口痰用时（1.73±0.81）h ，较对照组（3.02±0.88）h 缩短，差异有统计学意义（t =3.447，P ＜0.01）。
观察组肺部并发症发生率低于对照组，差异有统计学意义（P ＜0.01）。
观察组入院及术后第3天晨起的PEF 差值的秩均值小于对照组（P ＜0.05）。
两组均未出现严重或者影响机体状态的不良事件。
结论 指压天突穴引咳法在PEF ≤5.33L/s 的早期肺癌术后患者咳嗽排痰中应用效果明显。
关键词： 穴位按压； 天突穴； 早期肺癌； 术后并发症； 排痰中图分类号： R 473.6 文献标志码： A 文章编号： 2709-1961（2023）11-0130-06Application of finger -pressing on Tiantu acupoint to inducecough in postoperative patients with early -stage lung cancerBIAN Guanjun 1，2，ZHANG Chunmei 3，LIU Lifeng 2（1. Tianjin University of Traditional Chinese Medicine ， Tianjin ， 301617；2. Department of Lung Cancer ， Tianjin Medical University Cancer Institute and Hospital ，National Clinical Research Center for Cancer ， Tianjin Key Laboratory for Cancer Prevention and Treatment ，Tianjin Cancer Clinical Research Center ， Tianjin ， 300060；3. School of Nursing Tianjin University of Traditional Chinese Medicine ， Tianjin ， 301617）ABSTRACT ： Objective To investigate the application of finger -pressing on Tiantu acupoint toinduce cough in postoperative patients with early -stage lung cancer.Methods Totally 100 patients with peak expiratory flow （PEF ） ≤5.33L/s who were admitted to the Hospital from January 2023 to March 2023 were randomly divided into observation group and control group ， with 50 cases in each group. The control group received conventional nursing measures ， while the observation group re⁃ceived finger -pressing therapy on Tiantu acupoint based on the basis of the control group. The time taken to cough up the first sputum after nebulization on the first postoperative day ， the difference of Peak Expiratory Flow （PEF ） on first day of hospital admission and three days after surgery ， post⁃operative pulmonary complications and intervention effect on coughing were compared between the two groups.Results The rate of effective cough -inducing in the observation group was significantly higher than that of the control group （P ＜0.01）. The time （h ） to cough up the first sputum after nebulization on the first postoperative day was shorter in the observation group （1.73±0.81） than inDOI ： 10.55111/j.issn 2709-1961.202307028· 中医特色护理 ·收稿日期：2023 - 07 - 28基金项目：天津市医学重点学科（专科）建设项目（TJYXZDXK -011A ）通信作者：张春梅， E -mail ： 2416353567@··130Vol.9， No.11，2023Chinese Journal of Integrative Nursingthe control group （3.02±0.88）， with a significant difference（P＜0.01）. The incidence of postop⁃erative pulmonary complications in the observation group was lower than that in the control group （P ＜0.01）. The rank of the difference of PEF on first day of hospital admission and three days after surgery was lower in the observation group than that in the control gorup（P＜0.05）. Neither of the groups reported adverse reactions.Conclusion Finger-pressing therapy on Tiantu acupoint to induce coughing is effective in coughing and sputum evacuation in early-stage lung cancer patients with PEF ≤5.33 L/s， and reduces pulmonary complications.KEY WORDS：acupressure；Tiantu acupoint；early-stage lung cancer；postoperative complications肺癌是我国死亡率最高的恶性肿瘤［1］，手术是早期肺癌治疗的主要方法［2］。
Angiogenesis is the outgrowth and proliferation of capil-laries from pre-existing blood vessels. This process is dis-tinct from the other major form of neovascularization, vasculogenesis, in which new blood vessels are formed from endothelial precursor cells1–3. During angiogenesis, capillary sprouts are generated after protease-mediated degradation of the endothelial basement membrane, which enables migration of endothelial cells into the surrounding interstitial tissue1,3,4. Stromal cells such as fibroblast-like synoviocytes (FLSs) and immune cells infiltrating the synovial tissue are the main source of these proteases, as well as of several other proangiogenic mediators5. Furthermore, pericytes have an important role in both initiation of angiogenesis and the matura-tion of vessels. Pericytes are accessory cells that surround the vascular endothelium; they are instrumental in reg-ulating blood vessel diameter, vascular permeability and endothelial cell proliferation, owing to their involvement in key processes in angiogenesis, such as lumen forma-tion, anastomosis of newly formed blood capillaries and the deposition of new basement membrane1,4,6. Pericytes also have a role in leukocyte recruitment and affect endothelial cell migration by altering the extra c ellular matrix7. The formation of new blood vessels results in an increased endothelial surface area, which further facili-tates inflammatory cell migration into the inflamed tissue through vasodilatation and increased endothelial cell permeability. Mediators involved in this mechanism include histamine, 5-hydroxytryptamine, leukotrienes, complement proteins and platelet-activating factor3,8. Angiogenesis is involved in several physiological and pathological processes, including cancer and chronic inflammation1,3. In 1971, researchers postulated that tumour growth is largely dependent on the neovascu-larization of tumour tissues9, and this hypothesis ush-ered in an era of intense research into the subject. Now, mounting evidence supports the involvement of on g oing angiogenesis in chronic inflammation and synovial pan-nus formation, both of which processes are involved in various arthritides2,3. Several proangiogenic signalling pathways that are activated in inflammation overlap with those that drive tumour angiogenesis. Hence, important insight into the processes underlying neovascularization in chronic inflammation can be gained from oncolog-ical studies, especially with respect to antiangiogenic therapy — which is nowadays a standard addition to treatment regimens for many types of cancer.Even though a wide range of molecules can induce angiogenesis under either physiological or patholog-ical conditions, the intracellular signalling pathways activated by such mediators are largely the same, and often overlap. In this Review, we discuss the most rele-vant molecules and signalling pathways involved in the above-mentioned angiogenic mechanisms. We also discuss emerging data on the regulation of these signal transduction mechanisms in chronic inflammation, and findings from preclinical and clinical trials of agents tar-geting these pathways, which can block angiogenesis and consequently ameliorate inflammation.Molecular mechanisms of angiogenesis Angiogenesis occurs via a complex series of processes involving numerous molecular mechanisms that mod-ulate neovascularization in inflammation (FIG. 1). Many of these processes are closely linked and activation of one can induce or alter the activity of another. In pathological1Amsterdam Rheumatology & Immunology Centre, Department of Experimental Immunology, Academic Medical Centre and University of Amsterdam, EULAR & FOCIS (Federation of Clinical Immunology Societies) Centre of Excellence, Meibergdreef 9, F4‑105, 1105 AZ Amsterdam, Netherlands.2Department of Rheumatology, Instituteof Medicine, University of Debrecen, Facultyof Medicine, Nagyerdei Str. 98, Debrecen 4032, Hungary. Correspondence to S.W.T. (s.w.tas@amc.uva.nl).doi:10.1038/nrrheum.2015.164 Published online 3 Dec 2015T argeting of proangiogenic signalling pathways in chronic inflammationSander W. T as1, Chrissta X. Maracle1, Emese Balogh2 and Zoltán Szekanecz2Abstract | Angiogenesis is de novo capillary outgrowth from pre-existing blood vessels. This process not only is crucial for normal development, but also has an important role in supplying oxygen and nutrients to inflamed tissues, as well as in facilitating the migration of inflammatory cells to the synovium in rheumatoid arthritis, spondyloarthritis and other systemic autoimmune diseases. Neovascularization is dependent on the balance of proangiogenic and antiangiogenic mediators, including growth factors, cytokines, chemokines, cell adhesion molecules and matrix metalloproteinases. This Review describes the various intracellular signalling pathways that govern these angiogenic processes and discusses potential approaches to interfere with pathological angiogenesis, and thereby ameliorate inflammatory disease, by targetingthese pathways.NATURE REVIEWS |RHEUMATOLOGY ADVANCE ONLINE PUBLICATION |1REVIEWSangiogenesis, resolution of these processes is generally lacking, which leads to continuous activation of several of these mechanisms.Hypoxia and the VEGF pathwayOne of the most critical triggers of angiogenesis is hypoxia. When oxygen availability in a tissue drops suffi c iently, oxygen-sensing prolyl hydroxylases become activated and hydroxylate the hypoxia-inducible factor (HIF) proteins, HIF-1 and HIF-2. These proteins form heterodimers that translocate to the nucleus where transcription of genes containing HIF-responsive ele-ments occurs. Although many of the downstream effects of the prolyl hydroxylase–HIF pathway — such as increased expression of VEGF (vascular endothelial growth factor) — are linked to angiogenic processes, it is important to note that this pathway is also actively involved in inflammation. HIF proteins not only interact with members of the nuclear factor κB (NFκB) family of transcription factors, which are discussed in further10.in inflammatory arthritis, and the role of11,12. Although VEGF is the main growth factor3,6.2,3,13–15. Interactions between VEGF,. Interestingly, the number of pericyte-lined(RA) than in those with osteoarthritis (OA) or healthy indivi d uals. Vessel stabilization and the elimination of immature vessels, or both, might be of therapeutic inter-est in RA. Interestingly, vessel stabilization is one of the consequences of anti-TNF therapy in RA17.Tissue remodellingAs mentioned above, remodelling of the surround-ing tissue is a critical step during neovascularization. Extracellular matrix components, adhesion recep-tors and proteases are involved in this remodelling and in perpetuation of angiogenesis in arthritis. The thrombospondin-1–TGF-β–CTGF axis is a recog-nized pathway in inflammatory angiogenesis and might also contribute to RA-associated cardiovascular disease18. Signalling through various pathways invol-ving integrins (such as αvβ3), their ligands, E-selectin, and focal adhesion kinases (FAKs) are important ininfiltration and fibroblast proliferation leads to an increase in cell populations, causing the microenvironment to become hypoxic. Hypoxia induces the production of proangiogenic factors (such as growth factors and matrix remodelling enzymes) by several cell types in the inflamed tissue. Binding of proinflammatory mediators such as cytokines and chemokines to cell surface receptors activates intracellular signalling pathways (inset), which can also have proangiogenic effects, further driving neovascularization as well as inflammation. PDGF, platelet-derived growth factor; R, receptor; VEGF, vascular endothelial growth factor. Adapted with permission from NPG © Cristofanilli, M. et al. Nat. Rev. Drug Discov.1, 415–426 (2002).angiogenesis 2,3,19–21. The activation of junctional adhesion molecules and synovial-tissue-degrading enzymes, such as matrix metalloproteinases (MMPs), is a feature of inflammatory arthritis and also has crucial roles during neovascularization 2,3,22–24.Proinflammatory cytokines and chemokinesAlthough hypoxia is considered to be the main driver of angiogenesis, inflammation also makes important contributions to this process. Several proinflamma-tory cytokines are considered to be proangiogenic, including TNF, IL-1, IL-6, IL-8 (also known as CXC motif chemokine 8 (CXCL8)), IL-15, IL-17, IL-18, G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte–macrophage colony-stimulating factor), and oncostatin M 2,3,25. These factors either exhibit direct proangiogenic activity or act indirectly via VEGF-dependent pathways. For example, TNF induces VEGF production in several cell types, including endothelial cells and macrophages 26,27. These inflamma-tory mol e cules also upregulate the expression of other angiogenic mediators, including adhesion molecules and MMPs, and can also both activate and inhibit the ANG–TIE-2 signalling axis 28,29.Chemokines also have a critical role in inflammation- induced angiogenesis, and a number of CXC and CC chemokines induce angiogenesis in RA 30. The proan-giogenic nature of many CXC motif chemokines is associated with a Glu–Leu–Arg (ELR) amino acid motif within their structure; ELR-containing proangio-genic CXC chemokines include CXCL1 (also known as growth-regulated α protein), CXCL5, CXCL6 and CXCL8. However, several CXC chemokines lacking the ELR motif have dual roles (that is, both proangiogenic and angiostatic) in neovascularization. For example, the CXCL12–CXCR4 (stromal cell-derived factor 1–CXC chemokine receptor type 4) axis is fundamentally important in both angiogenesis and vasculogenesis as it attracts endothelial precursor cells to line the newly formed blood vessels 31,32. In a mouse model of arthritis, CXCL16 was involved in both attraction of endothelial precursor cells and synovial angiogenesis 33. Conversely, the ELR-lacking chemokines CXCL4 (also known as platelet factor 4), CXCL4L1 (also known as platelet fac-tor 4 variant), CXCL9, CXCL10, CXCL11, CXCL13 and CXCL14 exert angiostatic effects in several models of tumour angiogenesis 34. Of note, CXCL9, CXCL10 and CXCL11 contain IFN-γ-inducible gene elements that might regulate their angiostatic function, as IFN-γ itself has been demonstrated to be involved in angiostasis in several tumour models 35,36.Chemokines can also indirectly promote angio-genesis through attracting immune cells to sites of inflammation, which induces further neovasculari-zation. For example, CCL2 (also known as monocyte chemo a ttractant protein 1) is considered important to angio g enesis because it attracts monocytes and macro-phages, which are a major source of proangiogenic mediators. In addition to its inherent ability to promote vessel formation, CX 3CL1 (also known as fractalkine) recruits leukocytes 37–39.Intracellular signalling pathwaysMany intracellular pathways that drive transcription of proinflammatory genes also influence angiogen e sis. In this section, we focus on the pathways that are best characterized in the context of chronic inflammation and hold potential for therapeutic targeting.NF κBOne of the most established signal transduction path-ways involved in inflammation is that involving NFκB. Five proteins make up the NFκB family, namely tran-scription factor p65, proto-onogene c-Rel, transcrip-tion factor RelB, and the NFκB p50/p105 and p52/p100 subunits (the cleaved forms of which, p50 and p52, are biologically active owing to their ability to form hetero-dimers with other NFκB members). These subunits form heterodimers that translocate to the nucleus where they activate gene transcription, leading to regulation of a multitude of biological functions, including cell pro-liferation, survival and migration, as well as activation of the immune response through the expression of pro-inflammatory cytokines and adhesion molecules. All these functions can contribute to neovascularization 40.NFκB signalling is subdivided into canonical and noncanonical pathways (FIG. 2). Canonical NF-κB sig-nalling is fast-acting, with changes in gene expression observed as early as 30 min after activation, and exerts its effects mainly through binding of p65–p50 hetero-dimers to DNA. By contrast, the slower non c anonical pathway requires ~4 h or more for changes in gene expression to occur, and is reliant on RelB–p52 hetero-dimers for gene transcription 40,41. Both pathways have been recognized for their proangiogenic potential in cancer as well as in many autoimmune (rheumatic) diseases 42. Signalling through the canonical pathway requires activation of the IKK (inhibitor of NFκB (IκB) kinase) complex and can be induced via a multitude of stimuli, for instance by Toll-like receptor ligands and proinflammatory cytokines such as TNF and IL-1β. In the case of TNF, induction of canonical NFκB signal-ling leads to VEGF production 26,27. Activation of NFκB via the canonical pathway also leads to the secretion of other proinflammatory molecules that can contribute to angiogenesis, such as TNF, IL-1β, IL-6, CCL2 and IL-8. Other proangiogenic factors produced through active canonical NFκB signalling include prostaglandin G/H synthase 2 (also known as cyclooxygenase-2), induci-ble nitric oxide synthase, MMPs and the adhesion mol-ecules ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion protein 1)28.The noncanonical pathway, which is induced via activation of TNF receptor superfamily members by stimuli such as lymphotoxin, TNF ligand superfam-ily member 14 (also known as LIGHT) and CD40 ligand, is also implicated in pathological angio-genesis associated with chronic inflammation and cancer. The central regulator of the noncanonical pathway, NIK (NFκB-inducing kinase, also known as MAP3K14 (mitogen-activated protein kinase (MAPK) kinase kinase 14)), is crucial to this process and mice lacking this protein exhibit remarkably lessR E V I E W Sneovascularization in the antigen-induced arthritis and B16-FL melanoma models43.MAPKsMAPK signalling represents another key axis in inflammation-induced angiogenesis that is sub d ivided into three distinct pathways, involving ERKs (extra-cellular signal-regulated kinases), JNKs (c-Jun N-terminal kinases) and p38 MAPKs, respectively (FIG. 2). Signal transduction through these pathways regu l ates vital bio-logical functions such as cell proliferation, differentiation and death44. The ERK MAPKs are activated primarily by growth factors, which stimulate cell growth and dif-ferentiation. JNK and p38 MAPK family members are activated by environmental stressors, such as ioni z ing radiation, heat, oxidative stress and DNA damage, as well as by inflammatory cytokines such as TNF, IL-1 and growth factors45.Mounting evidence demonstrates a crucial role for MAPK signalling in the pathogenesis of RA. Activation of all three families of kinases has been detected in RA synovial tissues and, of particular interest, expression of ERK MAPKs is mainly localized to synovial blood ves-sels46. In RA, activation of JNKs by IL-18 in FLSs leads to the secretion of VEGF, CCL2, and CXCL12. IL-18 also induces chemotaxis and tube formation by human micro-vascular endothelial cells (HuMVEC) and angio g enesis in Matrigel®(Discovery Labware Inc., USA) plugs, all of which are dependent on JNKs47. TNF can also signal through JNKs, leading to upregulation of ICAM-148. Activation of p38 MAPKs in endo t helial cells induces expression of the proangiogenic mol e cules E-selectin, VCAM-1 and CCL249, which all enhance immune cell infiltration in chronic inflammatory diseases, such as RA and inflammatory bowel disease (IBD)50.Protein C-ets-1 (also known as p54) is one of the transcription factors activated by the MAPK family of proteins, and is currently under investigation for its role in autoimmune diseases such as RA, as well as in can-cer51. C-ets-1 has an important role in several biological processes, including development, differentiation, pro-liferation, apoptosis, migration, tissue remodelling, cell invasion and angiogenesis52. For example, in endo t helial cells, C-ets-1 promotes angiogenesis via p38 MAPK signalling downstream of VEGF. In the chick chorio-allantoic membrane assay, C-ets-1 is involved in FGF-induced expression of MMP-1, as well as in endothelial cell migration52. C-ets-1 also regulates the production of proangiogenic molecules, such as TNF and CCL2, by lymphocytes53,54.PI3K–AKT (phosphatidylinositol-4,5-bisphosphate 3-kinase–RAC-α serine/threonine-protein kinase (also known as protein kinase B)). These pathways ultimately result in upregulation of processes that are central to angiogenesis, including cell growth, survival, migration, proliferation and differentiation. AP‑1, transcriptional activator protein 1; ERK, extracellular signal-regulated kinase; FOXO, forkhead box protein O; IκB, inhibitor of NFκB; IKK, IκB kinase; JNK, c-JunN‑terminal kinase; MAP2K, dual specificity MAPK kinase (also known as MEK); PTEN, phosphatase and tensin homologue; SOCS, suppressor of cytokine signalling.PI3K–AKTThe phosphoinositide 3-kinase (PI3K) pathway is another crucial signalling pathway involved in various cellular processes, such as cell growth, survival, migration, pro-liferation and differentiation. The PI3K family of kinases signals through AKT (RAC-α serine/threonine-protein kinase, also known as protein kinase B). AKT is essential for angiogenic processes in both physiological and patho-logical conditions (FIG. 2). In endothelial cells, the PI3K pathway is activated downstream of VEGF and probably influences the survival, growth and proliferation of these cells during neovascularization. AKT can also activate endothelial nitric oxide synthase in endothelial cells, as well as increase expression of both HIF-1α and HIF-2α, all central regulators of angiogenesis55,56. The adhesion molecule E-selectin also signals through the PI3K–AKT pathway and induces considerable increases in chemo-taxis and tube formation in HuMVEC57,58. These obser-vations might be relevant to the inflammation-induced angiogenesis associated with RA, as AKT expression in synovial tissue is significantly higher in RA than in OA59. In addition, the PI3K–AKT pathway is involved in microvascular dysfunction, which contributes to the pathogenesis of IBD60. Interestingly, mechanical strain in combination with proinflammatory signals results in increased PI3K-mediated proliferation of endothelial cells61. This mechanism might also be relevant to inflam-matory diseases, as endothelial cells experience strain during inflammation due to increased blood flow and oedema, in combination with tissue remodelling, which could be important in maintaining proangiogenic signals.JAK–STATThe Janus kinases (JAKs) are part of another important signalling pathway that influences cellular responses to inflammation. JAKs lie downstream of type I and II cytokine receptors and, upon activation, recruit sig-nal transducer and activators of transcription (STAT) proteins, which become phosphorylated and can then translocate to the nucleus where they activate transcrip-tion (FIG. 2). JAK–STAT signalling is negatively regu-lated by the SOCS (suppressors of cytokine signalling) family of proteins. STATs are established regulators of the genes encoding many proinflammatory molecules (including IL-17) and cellular adhesion molecules (such as ICAM-1)62. In RA, immunohistochemical studies have revealed increased expression of several activated STAT proteins in the inflamed synovium, implicating the JAK–STAT pathway in the pathogenesis of RA63–65. Furthermore, IL-6 in synovial fluid is the main activating factor of STAT3 and is also capable of activating STAT1 in synovial fluid neutrophils65–67. Of interest, genes impli-cated in JAK–STAT signalling as well as genes encoding proangiogenic chemokines were differentially expressed in inflamed colon tissue in both patients with IBD and a preclinical model of IBD68. Similar mechanisms might also occur in other chronic inflammatory diseases, such as RA. JAK–STAT signalling also has a role in angio-genesis, as GM-CSF activates both JAK2 and STAT3, leading to neovascularization in the chick chorioallantoic membrane assay69.FAKSignalling through FAKs is vital during physiological angiogenesis, and increasing evidence demonstrates that this family of kinases could also be an important contributor to pathological angiogenesis, specifically in chronic inflammatory diseases and cancer20,70,71. Activation of FAKs in endothelial cells occurs down-stream of receptors that are responsive to growth factors, integrins and cytokines; moreover, activated FAKs are crucial for endothelial cell migration, an indispensable aspect of angiogenesis (FIG. 2). FAKs also have a crucial role in cell adhesion, being the major signalling compo-nent acting downstream of integrin αVβ3. Levels of FAK expression are higher in patients with RA or OA than in healthy controls, as well as in patients with several types of cancer20. FAKs might also contribute to chronic intes-tinal inflammation as, in human intestinal microvascular endothelial cells, various bacterial ligands induce angio-genic responses via several signalling pathways, including FAK signalling50. FAK deletion in mice injected with lung carcinoma cells results in marked attenuation of pro-angiogenic responses to VEGF, and the endothelial cells isolated from these mice exhibit decreased proliferation and increased apoptosis72.Src kinasesThe Src family of kinases modulate signal transduction cascades involved in several biological processes that are crucial to angiogenesis, such as cell-cycle control, cell adhesion, migration and proliferation. Src kinases are activated by various growth factors, and signal down-stream of receptor tyrosine kinases (FIG. 2). Specifically, Src kinases are activated by VEGF and bFGF (basic FGF) in endothelial cells; however, activation of Src kinases seems to be dispensable for bFGF-mediated cell growth73. Src kinases owe their involvement in several cellular pro-cesses to their ability to interact with members of various other pathways, including all the aforementioned path-ways described in this Review. For example, Src kinases, in combination with activated JNKs, play a central part in IL-18-induced angiogenesis in RA58. Src kinases can also signal in parallel with the PI3K pathway, and do so during angiogenesis induced by soluble E-selectin57.Sphingosine kinase 1Another emerging candidate of interest in pathological angiogenesis is sphingosine-1-phosphate (S1P), a bio-active lipid that participates in physiological processes such as cell growth, differentiation, migration, survival and angiogenesis74(FIG. 3). S1P is generated by sphingo-sine kinase 1 (SPK 1), which has been implicated in the regulation of TNF-dependent release of IL-1β and IL-6. Levels of S1P in synovial fluid and synovial tissue are higher in patients with RA than in those with OA75–77. Moreover, S1P and SPK 1 have been detected in the cytoplasm of vascular endothelial cells isolated from the inflamed salivary glands of patients with primary Sjögren syndrome78.Overexpression of SPK 1 in vascular endothe-lial cells results in enhanced migratory capacity and an increased rate of capillary tube formation76.R E V I E W SFurthermore, these endothelial cells show evidence of constitutive activation, specifically the induction of basal VCAM-1 expression and an augmented VCAM-1 and E-selectin response to TNF76. The resulting improve-ment in neutro p hil binding could be blocked by over-expression of a dominant-negative form of SPK 175–77,79. Moreover, S1P contributes to P-selectin-dependent leukocyte rolling (and consequently to chronic inflam-mation) through endothelial S1P receptor 3. S1P also regulates VEGF expression by articular chondro-cytes, and might, therefore, be indirectly important in arthritis-related angiogenesis80.NotchNotch signalling is an evolutionarily conserved path-way that is crucial to endothelial cell interactions and, consequently, also to angiogenesis81–84(FIG. 3). Five canonical ligands (delta-like proteins 1, 3 and 4, pro-tein jagged-1 and protein jagged-2) are expressed in mammalian cells and bind to the corresponding Notch receptors (Notch 1–4). Upon binding, the Notch recep-tor intra c ellular domain is cleaved through the actions of γ-secretase. Nuclear translocation of this cleaved domain leads to the transcription of genes important in angiogen-esis83. Notch ligands and receptors are highly expressed in endothelial cells as well as various tumour cells, in which Notch signalling is implicated in vessel matura-tion83,84. Furthermore, Notch ligands and receptors are highly expressed in the endothelial cells of patients with thromboangiitis obliterans (Buerger disease), a rare pro-gressive thrombotic vasculopathy characterized by non-atherosclerotic, inflammatory occlusion of segments of medium and small arteries and veins, especially of the hands and feet85. In RA, Notch signalling has been implicated in hypoxia-induced and VEGF-mediated angiogenesis, suggesting that this pathway could be an attractive therapeutic target in this disease81,82.Potential therapeutic approaches Approaches that target angiogenesis are part of a promising new era in the treatment of several condi-tions characterized by pathological angiogenesis, most importantly tumour growth in cancer and chronic inflammatory diseases such as RA. In this section, we highlight novel therapeutic agents that target the most prominent proinflammatory intracellular signallingpathway. However, S1P also has important intracellular (second messenger) actions and can interfere with TNF receptor signalling and histone deacetylases. S1P also affects cytoskeletal structure through Rho and Rac signalling, which is important for cell migration and angiogenesis. Notch signalling can be activated by ligands such as delta-like proteins 1 and 4 or jagged-1 and jagged-2. Subsequently, γ-secretase cleaves the Notch receptor intracellular domain (NICD) from the Notch receptor, enabling NICD to translocate to the nucleus where it interacts with RBPJ (recombining binding protein suppressor of hairless, also known as Jκ-recombination signal-binding protein), resulting in recruitment of additional co-activators, such as mastermind-like proteins (MAMLs), leading to the transcription of genes important in angiogenesis. IκB, inhibitor of NFκB; IKK, IκB kinase; TRAF, TNF receptor associated factor.pathways contributing to pathological angiogenesis 13,14 (TABLES 1–3).Tyrosine kinase inhibitors Several tyrosine kinase inhibitors are currently under investi g ation for their ability to block the intra c ellular signalling components downstream of major growth factor receptors involved in angiogenesis, such as VEGF receptors, predominantly in the setting of anti t umourtherapy. Sunitinib is a well-established receptor tyro-sine kinase inhibitor that originally entered the clinic as an antiangiogenic agent for the treatment of malignant tumours. However, this agent also inhibits synovial neo v ascularization in mice with collagen-induced arthritis, and this improvement is accompanied by a dose-dependent beneficial effect on clinical arthritis scores 86.Pazopanib, a multitargeted tyrosine kinase inhibitor that blocks the VEGF receptor as well as PDGF receptors α and β, was also effective in inhibiting angiogenesis both in vitro and in vivo in phase II clinical trials for treatment of renal cell carcinoma and other solid tumours 87–90. This agent has been approved by the FDA for the treatment of renal cell and soft tissue carcinomas 90. The FGF-1 receptor tyrosine inhibitor PD166866 inhibits micro-vessel outgrowth in human placental artery fragments, demonstrating its therapeutic potential for blocking pathological angiogenesis 91. The therapeutic potential of pazopanib and PD166866 in chronic inflammation remains to be tested.NF κB signalling inhibitors Owing to the importance of NFκB signalling in regu-lating inflammation, more than 700 compounds with reported inhibitory effects on NFκB signalling have been developed 14,92. Strategies targeting the canonical NFκB pathway that have been tested in preclinical mod-els of inflammation include small-molecule inhibitors 92, NEMO (NFκB essential modulator)-binding domain peptides that specifically interfere with activation of the IKK complex 93, and intra-articular gene transfer of a dominant-negative form of IKKβ94. However, no clini-cal trials of these approaches have been conducted, most likely owing to concerns about the potential for off-target effects and toxicity associated with global NFκB inhi-bition, considering the importance of this pathway incellular homeostasis.A potential solution would be to develop approachesthat target NFκB signalling in endothelial cells only.For example, a multimodular recombinant protein, SLC1, has been developed that specifically binds tocytokine-activated endothelial cells and delivers a NEMO-binding domain peptide specifically to these cells. After delivery, this peptide disrupts the IKK com-plex, resulting in reduced leukocyte–endothelium inter-actions of trafficking immune cells, and potentiallyalso inhibition of angiogenesis. Treatment with thisagent resulted in decreased inflammation, as demon-strated by the compound’s ability to ameliorate bothserum-transfer-induced and antigen-induced arthritis in mice 95.As mentioned previously, activation of the non-canonical NFκB pathway in endothelial cells also has animportant role in pathological angiogenesis associatedwith arthritis. Targeting of this pathway rather than the canonical pathway might result in a reduced severity of adverse effects, and is currently being tested in preclinical models using small-molecule inhibitors of NIK, the main regulating kinase of the noncanonical NFκB pathway.MAPK inhibitorsSimultaneous targeting of all three members of the MAPK family is also currently being studied as a possible mechanism to block pathological angiogenesis. One such nonspecific inhibitor, tacrolimus, was demonstrated to inhibit the production of IL-1β-induced ANG-1, TIE-2 and VEGF expression by FLSs through downregulation of JNKs and p38 MAPKs, thereby probably indirectly suppressing angiogenic responses in the inflamed syno-vium 96. An ERK inhibitor, FR180204, has shown efficacy in the collagen-induced arthritis mouse model by signifi-cantly reducing clinical arthritis scores after preventative administration 97. The JNK-specific inhibitor, SP600125, inhibited microvascular endo t helial cell proliferation and migration as well as inhibiting tumour growth in a human prostate carcinoma xenograft model 98. This compound also decreased paw swelling in the rat adjuvant-induced arthritis model, and the clinical score improvement in treated rats was accompanied by a substantial reduction of radiographic damage 99. At present, several companies are developing JNK inhibitors, but no data have yet beenreported from clinical trials of these agents in patientswith arthritis or other inflammatory diseases.Inhibitors of p38 MAPKs also show promise fortargeting angiogenesis. The p38-selective inhibitor ral-imetinib reduced endothelial cord formation driven by VEGF, bFGF, EGF or IL-6 as well as by tumour cells in both in vitro and in vivo cancer models, demonstrating its possible efficacy in targeting tumour angiogenesis 100. Other p38-MAPK-specific small-molecule inhibitors have been developed; however, despite demonstrating potent antiangiogenic effects in animal studies, clinical trials of these agents in patients with RA or IBD have failed owing to lack of efficacy or toxic effects (most notably elevated levels of liver enzymes)101–104. A possible Table 1 |FDA-approved compounds that target angiogenesis pathwaysR E V I E W S。
