骨修复Novel therapeutic core–shell hydrogel scaffolds with sequential delivery of cobalt and bone

The International Journal of Biochemistry&Cell Biology38(2006)671–683Overexpression of the novel human gene,nuclearapoptosis-inducing factor1,induces apoptosisBingfeng Lv a,b,1,Taiping Shi c,1,Xinyu Wang c,Quansheng Song a,Yingmei Zhang b,Yan Shen c,d,Dalong Ma a,b,c,Yaxin Lou b,c,d,∗a Lab of Medical Immunology,School of Basic Medical Science,Health Science Center,Peking University,38Xueyuan Road,Beijing100083,Chinab Center for Human Disease Genomics,Peking University,38Xueyuan Road,Beijing100083,Chinac Chinese National Center of Human Genome Research(Beijing),3North Yongchang Road,BDA,Beijing100176,Chinad National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences/Peking Union Medical College,Beijing100730,ChinaReceived8August2005;received in revised form11November2005;accepted14November2005Available online9December2005AbstractApoptosis is a genetically determined cell suicidal program that plays critical roles in many physiological and pathological processes.In this study,we report the cloning and characterization of a novel human gene,nuclear apoptosis-inducing factor1 (NAIF1),overexpression of which induces apoptosis in cells.Human NAIF1is located on chromosome9q34.11and encodes327 amino acids with a homeodomain-like region and two nuclear localization signals at its N-terminal region.NAIF1is conserved across diverse species,including human,mouse,crab-eating macaque,dog,chicken and frog,and shares no obvious homology to any known genes or proteins.Northern blot analysis revealed wide expression of NAIF1mRNA throughout human tissues.NAIF1 was predominantly localized in the nucleus.Overexpression of NAIF1inhibited cell growth and induced apoptosis.Furthermore, NAIF1transfection caused both decreases in mitochondrial membrane potential and caspase-3activation.In summary,NAIF1is a nuclear protein that induces apoptosis when overexpressed.©2005Elsevier Ltd.All rights reserved.Keywords:Nuclear protein;Programmed cell death;Nuclear apoptosis-inducing factor1;C9orf90;Homeodomain-like region1.IntroductionApoptosis is a genetically determined cell suicidal program that eliminates unwanted,redundant and dam-Abbreviations:AIF,apoptosis-inducing factor;AIFL,apoptosis-inducing factor like;C9orf90,chromosome9open reading frame 90;NAIF1,nuclear apoptosis-inducing factor1;ORF,open reading frames;PMSF,phenylmethylsulfonylfluoride∗Corresponding author.Tel.:+861082801149;fax:+861082801149.E-mail address:louyaxin@(Y.Lou).1These authors contributed equally to this work.aged cells,and it is required for maintaining a bal-ance between cell proliferation and cell death(Kerr, Winterford,&Harmon,1994).Apoptosis is essential in sculpting the developing organism during embry-onic and fetal growth(Meier,Finch,&Evan,2000), and is critical in the adult for maintaining the home-ostasis of differentiated tissues(Hengartner,2000).This type of cell death is characterized by a biochemi-cal cascade,which involves activation of caspases and translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane,and by distinct morphological changes including blebbing of cytoplas-1357-2725/$–see front matter©2005Elsevier Ltd.All rights reserved. doi:10.1016/j.biocel.2005.11.007672 B.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–683mic membranes,chromatin condensation,margination and fragmentation into apoptotic bodies(Hengartner, 2000;Kerr,Wyllie,&Currie,1972;Thornberry& Lazebnik,1998).A variety of stimuli,including of growth factor deprivation,signaling via certain sur-face receptors,treatment with DNA-damaging agents and inhibitors of macromolecular synthesis,can induce apoptosis via receptor-mediated or mitochondrial path-ways(Ellis,Yuan,&Horvitz,1991;Steller,1995).Dys-regulation of apoptosis has been associated with many serious human diseases,including neoplasia,degener-ative disorders and autoimmune diseases(Nicholson, 2000;Zimmermann,Bonzon,&Green,2001).Thus, studies on apoptosis are of great value for understanding events that occur during development as well as in the adult.By searching the human Refseq and expressed sequence tag(EST)databases,we obtained sequences of hundreds of novel human genes,whose open read-ing frames(ORF)were longer than300base pairs.The ORFs of these genes were cloned into pcDNA3.1-myc-his B vectors and underwent several screening analyses. By observing the morphological changes in transfected cells,we found that a novel human gene,NAIF1(also named C9orf90)caused cells to undergo marked round-ing and eventual detachment from the culture dish.The results of database searching suggested that NAIF1is conserved among human,mouse,crab-eating macaque, dog,chicken and frog,and shared no obvious homology to any known genes or proteins.Northern blot analy-sis revealed ubiquitous expression of NAIF1through-out human tissues and subcellular localization analysis revealed that NAIF1was located in the nucleus.Fur-ther studies indicated that overexpression of NAIF1 inhibited cell growth and survival,while also induc-ing cell shrinkage and chromatin condensation,both of which are hallmarks of apoptosis.Finally,we found that NAIF1caused depolarization of mitochondrial mem-brane potential and caspase-3activation in HeLa cells. Thus,in the present study,we cloned the NAIF1gene and found it to induce apoptosis when overexpressed in cells.2.Materials and methods2.1.MaterialsRPMI1640and DMEM,fetal bovine serum(FBS) and penicillin-streptomycin were purchased from Invit-rogen(USA).MTT[3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide],monoclonal antibod-ies against␤-actin and FLAG-tag were purchased from Sigma-Aldrich(St.Louis,USA).The cationic lypophilicfluorochrome3,3 -dihexyloxacarbocyanine iodide[DiOC6(3)]was from Molecular Probes(USA). Monoclonal antibodies against caspase-3and PARP were obtained from Transduction Laboratories(Becton Dickinson,USA).2.2.Cell culture and cell linesHeLa cells,a human cervical carcinoma cell line, were maintained in DMEM supplemented with10% heat-inactivated FBS,100U/ml penicillin and100␮g/ml streptomycin.Human embryonic kidney cells,HEK293, were maintained in RPMI1640medium supplemented as DMEM.Cells were grown at37◦C in a humidified5% CO2atmosphere and used for assays during exponential phase of growth.2.3.Northern blot analysisA470-bp PCR product corresponding to513–982bp of NAIF1coding region was used as a probe and labeled with␣-32P-dCTP using a Random Primers DNA Label-ing System(Invitrogen,USA)as described by the man-ufacturer.Human multiple tissue Northern blots(Clon-tech,USA)were hybridized with the probe and washed according to the procedure provided by the manufacturer. The membranes were exposed to a phosphor-imaging screen overnight and analyzed using the Cyclone TM Stor-age Phosphor System(Packard Instrument Company, USA).2.4.Cloning,plasmids construction and transfectionThe complete coding region of NAIF1was ampli-fied from a human liver cDNA library(Clontech,USA) by PCR using the forward primer P1(5 -GGG GGT GGG TGA GGG GC-3 )and reverse primer P2(5 -ACC CCT GCC CTC ACT GGA TG-3 ).The cDNA encod-ing amino acids71–327of NAIF1,NAIF1(71–327), was amplified using P3(5 -CGA ATT CCA CCA TGT GGT CTG ACC TCA AGA CCG A-3 )and P2as primers.The PCR products were cloned into pGEM-T Easy vector(Promega,USA)and sequenced using ABI PRISM®3100Genetic Analyzer(Applied Biosystems, USA).The inserts were released by cutting with Eco RI and subcloned into the Eco RI site of the mammalian expression vector pcDNA.3.1-myc-His B(Invitrogen, USA).To create the expression vectors carrying GFP at the N-terminal ends of NAIF1or NAIF1(71–327),theB.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–683673ORF of NAIF1and NAIF1(71–327)were subcloned in frame into pEGFP-N1(Clontech,USA).To con-struct a Flag-tagged NAIF1expression vector,the ORF of NAIF1was subcloned in frame into the pFLAG-CMV vector(Sigma-Aldrich,St.Louis,USA),which allowed for expression of NAIF1carrying Flag-tag at its N-terminal in mammalian cells.The resulting vectors,named pEGFP-N1-NAIF1,pEGFP-N1-NAIF1 (71–327)and pFlag-NAIF1were sequenced to confirm correct in-frame fusion between GFP or Flag-tag and NAIF1.The Bax expressing vector was described previ-ously(Wang et al.,2004).DNA transfections were performed using Lipofec-tamine2000(Invitrogen,USA)according to the man-ufacturer’s instruction or by electroporation at a single pulse of120V,20ms,with10␮g plasmid per106cells in2mm gap cuvettes using an ECM830square wave electroporation system(BTX,USA).2.5.Protein extraction and immunoblottingCells were pelleted by centrifugation,lysed in lysis buffer(20mM Tris–HCl,pH7.4,150mM NaCl,1mM EDTA,1mM EGTA,1mM PMSF)and incubated for 30min on ice.Lysates were centrifuged at18,000×g for10min at4◦C and the supernatant was mea-sured using the BCA protein assay reagent(Pierce, USA).Equal amounts of protein(20␮g)were separated by sodium dodecyl sulfate/polyacrylamide gel elec-trophoresis(SDS-PAGE)and transferred onto nitrocel-lulose membranes(Amersham Pharmacia,UK).Mem-branes were blocked in Tris-buffered saline containing 0.1%Tween-20(TBS-T)and5%non-fat milk for2h and incubated overnight at4◦C with the appropriate primary antibodies.Blots were washed three times for 10min each with TBS-T and incubated for1h in the dark with the appropriate IRDye TM800-conjugated secondary antibodies(LI-COR Bioscience Inc.,USA)prepared in TBST/5%non-fat milk.The signal was detected using the Odyssey®Imaging System(LI-COR Bioscience Inc.,USA).2.6.MTT assay and colony forming assayFor MTT colorimetric assay,the transfected cells were placed in96-well culture dishes at a density of 2000cells/well in100␮l medium.At the indicated time points,MTT solution was added and samples were incu-bated for4–6h.The formazan dye was then solubilized and the absorbance was measured at570nm.A colony forming assay was performed as described previously (Wang et al.,2004).Briefly,HeLa and HEK293cells were trypsinized24h post-transfection and a total of 1000cells were seeded in each100mm tissue culture dish.Selection medium with G418(800␮g/ml)was added.Three weeks later,colonies werefixed and stained with crystal violet.Colonies with a diameter of more than0.5mm were counted.Each group was assayed in triplicate and each experiment was repeated three times.2.7.Detection of phosphatidylserine externalizationTransfected HeLa cells(2×105)were trypsinized, washed twice with PBS,and resuspended in200␮l bind-ing buffer(10mM HEPES,pH7.4,140mM NaCl,1mM MgCl2,5mM KCl,2.5mM CaCl2).FITC-conjugated Annexin V(Biosea,China)was added to afinal con-centration of0.5␮g/ml.After incubation for20min at room temperature,propidium iodide was added to afinal concentration of1␮g/ml and the samples were analyzed on a FACSCaliburflow cytometer(Becton Dickinson, USA)with excitation using a488nm argon ion laser.2.8.Measurement of mitochondrial membrane potentialDiOC6(3)was used to evaluate whether NAIF1-induced apoptosis was associated with the disruption of transmembrane potential in mitochondria.Transfected cells were harvested at the indicated time points and cells were incubated with standard culture medium containing 40␮M DiOC6(3)for30min.Subsequently,cells were washed with standard culture medium and immediately analyzed byflow cytometric analysis with a FACSCal-iburflow cytometer(Becton Dickinson,USA).2.9.Evaluation of caspase-3activity(DEVDase activity analysis)HeLa cells were transfected with empty vector, NAIF1or Bax expression vectors and harvested in lysis buffer(10mM Tris–HCl,pH7.5,10mM NaHPO4, 130mM NaCl,1%Triton-X100,1mM PMSF).Cell lysates were clarified by centrifugation at18,000×g for20min at4◦C.Cell lysates containing10␮g pro-tein were incubated at37◦C in buffer containing25mM HEPES,pH7.5,1mM EDTA,100mM NaCl,0.1% CHAPS and10mM dithiothreitol with thefluoro-genic substrate Ac-DEVD-AMC(ALEXIS Biochem-icals,Switzerland).To measure this signal,we used a FLUOStarfluorometer(BMG Labtechnologies,Ger-many)with an excitationfilter of380nm and emission filter of460nm.Results were calculated as a proportion674 B.Lv et al./The International Journal of Biochemistry &Cell Biology 38(2006)671–683of the control over 90min (T90to T0).Samples were prepared in triplicate.2.10.Statistical analysisResults were expressed as means ±standard devia-tions.Student’s t -test was used for statistical comparison.p values of less than 0.01were considered statistically significant.3.Results3.1.Cloning of NAIF1cDNAUsing functional screening of novel human cDNA,we found that a hypothetical gene,chromosome 9open read-ing frame 90(C9orf90),could induce apoptosis.To our knowledge,no functional study has been performed on this hypothetical gene.We named it nuclear apoptosis-inducing factor 1(NAIF1)because it was a nuclear protein that induced apoptosis when overexpressed in cells.The cDNA of NAIF1(Refseq number is NM 197956)is 3198bp long,containing an ORF encoding 327amino acids with an ATG initiation codon whose surrounding sequence (GCCATGG)matches Kozak’s rule (Kozak,1991).EST assembly and Ecgene database searching (http://genome.ewha.ac.kr/ECgene/)revealed that there were at least nine RNA splicing forms of human NAIF1,the length of which ranged from 697to 3406base pair.These nine variants of NAIF1encoded five different proteins,among which the variant encoding 327amino acids was the longest.In this study,NAIF1referred to the 327amino acids variant.We designed primers to amplify the coding region of NAIF1from human cDNAs.The resultant RT-PCR products were in accor-dance with the predicted size (data not shown).The band was purified and cloned into the pGEM-T Easy vector.The insert was sequenced and was identical to the coding region of the hypothetical gene C9orf90.The cDNA sequence of the human NAIF1coding region and predicted amino acid sequence are shown in Fig.1.Alignment of the putative NAIF1cDNA to human genomic sequences revealed that NAIF1is a single copy gene located on chromosome 9q34.11.Database search-ing indicated that NAIF1was conserved among human,mouse,dog,crab-eating macaque,chicken and frog,but shared no obvious homology to any known genes or proteins.The deduced amino-acid sequences of human,mouse,dog,crab-eating macaque and chicken NAIF1proteins were aligned in Fig.2A and thepercentagesFig.1.The cDNA and predicted amino acid sequences of human NAIF1.The predicted glycine-rich region (GRR)is underlined and the predicted NLSs are shown in bold.B.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–683675Fig.2.NAIF1is conserved across diverse species.(A)Amino-acid sequence alignment of NAIF1proteins of different species.Sequence alignment was obtained using the ClustalW algorithm(Slow\Accurate,Identity)of the Lasergene software(DNASTAR Inc.,Madison,WI).The boxed areas indicate matching residues.(B)Sequence comparison of NAIF1proteins.The numbers represent the percentage of sequence identity and divergency determined by the ClustalW method with Slow\Accurate,Identity weight table in the Lasergene software.676 B.Lv et al./The International Journal of Biochemistry &Cell Biology 38(2006)671–683Fig.3.Northern blot analyses.Human multiple tissue Northern blots were hybridized with the ␣-32P-labeled NAIF1probes.The RNA markers are indicated in kilobases.of identity between different species of NAIF1were shown in Fig.2B.PSORT II (http://psort.nibb.ac.jp/)software analysis indicated that there were two NLSs at the N-terminal in the NAIF1protein.Motif analy-sis by Prosite (/prosite/)suggested that there was a glycine-rich region (GRR)from amino acid 92–117of human NAIF1and InterProScan search (/InterProScan/)indicated the 84amino acids of the N-terminal of human NAIF1protein contained a homeodomain-like region,which may bind to DNA (Mannervik,1999).3.2.Northern blot analysesNorthern blot analyses were used to confirm the existence of NAIF1mRNA in human tissues.Human multiple tissue Northern blots were probed with a ␣-32P-labeled probe corresponding to 513–982bp of the human NAIF1coding region.As shown in Fig.3,more than one band was detected in almost all tissues stud-ied,indicating that there were alternative RNA splic-ing forms of human NAIF1in most human tissues,which was in accordance with the result of database searching.3.3.Transfected NAIF1is expressed in human cells The coding region of human NAIF1cDNA was cloned into the pCMV-Flag mammalian expression vec-tor,which expressed Flag-tagged NAIF1protein in human cells.The vectors were transfected into HeLa cells and 24h later,whole cell lysates from transfectedcells were extracted and subjected to Western blot analy-sis.In the lane containing NAIF1-expressing cell lysates,there was one main 45kDa band,which was about 10kDa larger than the predicted size of the NAIF1fusion protein.There were also two weaker bands with molecu-lar masses of 30and 55kDa (Fig.4),indicating that there were different post-translation modifications of recom-binant NAIF1protein.In the lane containing the control vector,there was no obviousband.Fig.4.Transfected NAIF1is expressed in human cells.HeLa cells were transfected with mock or pFlag-NAIF1vectors.Twenty-four hours after transfection,cells were harvested and subjected to Western blot analysis using anti-flag-tag antibody.B.Lv et al./The International Journal of Biochemistry &Cell Biology 38(2006)671–683677Fig.5.Subcellular distribution of human NAIF1.HeLa cells were transfected with expression vectors carrying NAIF1–GFP or GFP using electro-poration.Cells were washed three times with PBS,fixed with 4%paraformaldehyde and incubated with DAPI to stain nuclei.Cells were analyzed with an Olympus IX 71fluorescent microscope and photographs were captured using an Alta U2digital camera (Apogee Instruments Inc.,NY ,USA).3.4.NAIF1protein is localized in the nucleus To determine subcellular localization of NAIF1,we generated a NAIF1–GFP fusion construct carrying GFP at the N-terminal of NAIF1.HeLa cells were tran-siently transfected with NAIF1–GFP fusion constructs or GFP vector.Twenty-four hours after transfection,the transfected cells were stained with DAPI and analyzed by fluorescence microscopy.An intense green fluores-cent signal was exclusively detected inside the nuclei in NAIF1–GFP expressing cells,while the green signal was seen diffusely throughout the cells which expressed only EGFP (Fig.5).Similar subcellular distribution was seen in other cell lines such as HEK293cells (data not shown).These results suggested that NAIF1was a nuclearprotein.Fig.6.Overexpression of NAIF1inhibits cell growth and survival.HeLa (A)and HEK293(B)cells were transiently transfected with the NAIF1expression plasmids and control plasmids.MTT assay (A and B)and colony formation assay (C)were used to determine the effect of overexpression of NAIF1on cell growth and survival.Data are presented as mean ±standard deviation (n =3).*means significantly different from mock-transfected cells.678 B.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–6833.5.Overexpression of NAIF1inhibits cell growth and survivalTo examine the function of NAIF1in cell growth,we analyzed the effect of its overexpression on cell growth using a MTT assay.HeLa and HEK293cells were tran-siently transfected with NAIF1-expressing or control plasmids.Fig.6A and B showed that overexpression of NAIF1significantly inhibited the growth of HeLa and HEK293cells.We also used a colony formation assay to evalu-ate the inhibitory effects of NAIF1on cellgrowth. Fig.7.Overexpression of NAIF1induces apoptosis.HeLa cells were transfected with control(A,D and G),NAIF1(B,E and H)or Bax(C,F and I)expressing vectors and stained with DAPI.Morphological changes of transfected cells(A–F)were observed under a microscope24h(A–C) or48h(D–F)post-transfection.Nuclear changes of transfected cells(G–I)were examined under afluorescent microscope48h post-transfection. Phosphatidylserine externalization of transfected cells was measured by Annexin V binding assay(J)at indicated time points.B.Lv et al./The International Journal of Biochemistry &Cell Biology 38(2006)671–683679HeLa and HEK293cells were transfected with the NAIF1-expressing plasmids containing a neomycin resistance gene.Twenty-four hours after transfection,G418was added and transfected cells were selected for 3weeks.Fig.6C showed that overexpression of NAIF1almost completely inhibited the formation of stable clones as compared with mock-transfected cells.Taken together with data from Fig.6A and B,these results suggested that NAIF1inhibited cell growth and stable colony formation in HeLa and HEK293cells.3.6.Overexpression of NAIF1induces apoptosis We observed morphological changes in transfected cells using fluorescence microscopy.As shown in Fig.7,there were no obvious morphological changes in NAIF1-or mock-transfected HeLa cells 24h post-transfection.However,Bax-transfected cells became round and detached from the cell culture plate within 24h.Forty-eight hours post-transfection,NAIF1-transfected cells underwent marked rounding and eventually detached from the culture dishes,similar to what had been observed in Bax-transfected cells.To confirm whether the NAIF1-transfected cells underwent apoptosis,we examined transfected cells stained with DAPI using fluorescence microscopy.Chromatin con-densation and margination,characteristic of apoptosis,were observed in NAIF1-and Bax-transfected cells 48h post-transfection.An Annexin V binding assay indicated that there were no obvious increases in the portion of Annexin V positive cells compared with mock-transfected cells 24h post-transfection,However,the number of Annexin V positive cells increased significantly in NAIF1-transfected cells,compared with mock-transfected cells 48h post-transfection (Fig.7J).These results indicated that NAIF1-transfected cells underwent an apoptotic process,but that NAIF1-induced apoptosis began much later than apoptosis induced by Bax.This phenomenon was not unique to HeLa cells.We also detected similar changes in HEK293cells (data notshown).Fig.8.Apoptosis of HeLa cells by overexpression of NAIF1is associated with a loss in mitochondrial transmembrane potential.Mitochondrial transmembrane potential ( Ψm )as reflected by DIOC6(3)staining of transfected HeLa cells was determined at indicated time points after trans-fection.Loss of Ψm was visualized as a reduction in the fluorescence signal in the FL1channel.The proportions of cells with reduced DIOC6(3)staining are shown.The results depicted are a representative of three independent experiments.680 B.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–6833.7.Overexpression of NAIF1disrupts mitochondrial transmembrane potentialThe disruption of mitochondrial transmembrane potential is one of the early features of apoptosis.There-fore,we evaluated whether NAIF1-mediated apoptosis was associated with this disruption.DiOC6(3),a cationic lypophilicfluorochrome,is commonly used for quantify-ing the mitochondrial transmembrane potential( Ψm) (Metivier et al.,1998;Petit,O’Connor,Grunwald,& Brown,1990).Transfected cells were pretreated with this dye and were analyzed byflow cytometry.Loss of DiOC6(3)staining indicates disruption of the mitochon-drial inner transmembrane potential( Ψm),which is associated with apoptosis(Susin et al.,1997).In com-parison with mock-transfected cells,NAIF1-transfected cells showed a decrease in their Ψm24and36h post-transfection as indicated by the percentage of cells with DiOC6(3)staining(Fig.8).DiOC6(3)staining was still reduced48h after transfection.Thesefindings suggested that NAIF1-induced apoptosis was associ-ated with a decrease in mitochondrial transmembrane potential.3.8.NAIF1activates caspase-3Activation of effector caspases,such as caspase-3and-7,is responsible for the proteolytic cleavage of a diverse range of structural and regulatory pro-teins in apoptosis(Salvesen&Dixit,1997).To deter-mine whether NAIF1activated caspase-3,we analyzed caspase-3activity in transfected cells using DEVD cleavage assay,a quantitative method used to detect caspase-3-like activity(Rehm et al.,2002).The results showed that DEVD cleavage activity in lysates from NAIF1-overexpressing HeLa cells were at leastfive times higher than mock-transfected cell lysates48h post-transfection(Fig.9A).To further confirm the activa-tion of caspase-3in lysates from NAIF1-overexpressing cells,we detected procaspase-3expression using West-ern blot analysis.We detected decreased procaspase-3 in lysates of NAIF1and Bax overexpressing cells com-pared to mock-transfected cells(Fig.9B).Because pro-teolytic cleavage of specific substrates by activated cas-pases is responsible for cellular dysfunction and struc-tural destruction associated with apoptosis(Thornberry &Lazebnik,1998),we analyzed the cleavage of PARP as a representative substrate in mock-,NAIF1-or Bax-transfected cells.PARP was cleaved in NAIF1-and Bax-transfected cells(Fig.9B).These results strongly suggested that the caspase cascade was activated during NAIF1-inducedapoptosis.Fig.9.NAIF1activates caspase-3.(A)DEVDase activity assay.HeLa cells were transfected with the empty vector or indicated constructs and,at indicated time,DEVDase activity was measured as described in Section2.Data are presented as mean±standard deviation(n=3). *means significantly different from mock-transfected cells.(B)Cellu-lar proteins were extracted from HeLa cells that were transfected with mock,NAIF1or Bax expressing plasmids.Protein levels were detected by western blot analysis with various antibodies as described in Section2.3.9.The N-terminal of human NAIF1is critical for nuclear localization and apoptosis-inducingfunctionComputer analysis indicated that there were two NLSs in the N-terminal of human NAIF1.To test whether this part was important for nuclear localiza-tion of NAIF1,plasmids expressing NAIF1–GFP or NAIF1(71–327)–GFP were transfected into HeLa cells. As shown in Fig.10A,NAIF1–GFP appeared as a nuclear protein,while NAIF1(71–327)–GFP was local-ized mainly in the cytoplasm.To determine whether the N-terminal of NAIF1was responsible for inducing apoptosis,we transfected plasmids expressing NAIF1 or NAIF1(71–327)mutant into HeLa cells.Forty-eight hours later,apoptosis was assessed by staining the nuclei of cells with DAPI.No nuclear morphological alterations were observed in mock-or NAIF1(71–327)-transfected cells.In contrast,NAIF1-transfected cells exhibited chromatin condensation and margination(Fig.10B).B.Lv et al./The International Journal of Biochemistry&Cell Biology38(2006)671–683681Fig.10.The N-terminal of human NAIF1is critical for nuclear localization and apoptosis-inducing function.(A)HeLa cells were transfected with NAIF1–GFP or NAIF1(71–327)–GFP vectors.Twenty-four hour post-transfection,cells werefixed and nuclei were stained with DAPI.(B)HeLa cells were transfected with mock,NAIF1or NAIF1(71–327)expression vectors.Forty-eight hours post-transfection,cells werefixed and nuclei were stained with DAPI.Thus,it appeared that the N-terminal of human NAIF1 was critical for its nuclear localization and apoptosis-inducing function.4.DiscussionIn this study,we cloned and characterized a novel human gene,NAIF1(also named C9orf90),whose over-expression induced apoptosis.NAIF1proteins are con-served in human,mouse,dog,crab-eating macaque, chicken and frog,indicating that it may have important functions in vertebrate animals.NAIF1shows no sig-nificant similarity to any known functional genes in the GenBank puter analysis suggested that the NAIF1protein may be localized in the nucleus.Con-sistent with the computer analysis,our results revealed that NAIF1was expressed mainly in the nucleus.The NAIF1(71–327)mutant was localized mainly in the cytoplasm and could not induce apoptosis,suggesting that amino acids1–70in the NAIF1protein may be an important region not only for apoptosis induction, but also for nuclear localization.It is also possible that NAIF1(71–327)did not induce apoptosis because of its localization in the cytosol,instead of the nucleus.Motif analysis indicated that there was a GRR from amino acid92–117of the NAIF1protein.GRR has been reported to be involved in important roles in cell function,such as RNA-binding,protein–protein interaction,tran-scriptional regulation,processing events,and nucleolar targeting(Gendra,Moreno,Alba,&Pages,2004;Nagao et al.,2000;Orian et al.,1999;Talbot et al.,1997;Yao, Yang,&Seto,2001).Recently,a study described that the GRR(aa152–227)in Sox4served as a novel func-tional domain for promoting apoptotic cell death(Hur et al.,2004).NAIF1may be another example that the GRR serves as a critical domain involved in apoptotic regulation.Further investigations are needed to identify the molecular mechanisms of NAIF1during apoptosis.Several different approaches have confirmed that NAIF1-induced apoptosis when overexpressed.First, NAIF1-transfected cells underwent marked rounding and chromatin condensation and margination(Fig.7). Second,NAIF1transfection increased the Annexin V positive cell population compared with mock-transfected cells(Fig.7).Third,NAIF1caused a depolar-ization in mitochondrial Ψm(Fig.8).Finally,NAIF1-induced caspase-3activation(Fig.9).Interestingly,all of these events appeared much later than in Bax-transfected cells.This delay could be due to the possibility that the apoptosis-inducing ability of NAIF1is simply weaker than that of Bax.Alternatively,the delay could be due to the possibility that NAIF1and Bax might induce apoptosis through different pathways,thus resulting in the induction of apoptosis on different time schedules.。

《中国组织工程研究》 Chinese Journal of Tissue Engineering Research文章编号:2095-4344(2018)18-02813-07 2813www.CRTER .org·研究原著·杜国庆，男，1990年生，山东省青岛市人，汉族，青岛大学口腔医学院在读硕士，主要从事正颌外科与组织工程骨方面的研究。

通讯作者：孙健，博士，教授，主任医师，硕士生导师，青岛大学附属医院口腔颌面外科，青岛大学口腔数字医学与3D 打印工程实验室，山东省青岛市 266003中图分类号:R318 文献标识码:A稿件接受：2018-05-11Du Guo-qing, Master candidate, Department of Oral and Maxillofacial Surgery, the Affiliated Hospital of QingdaoUniversity, Laboratory of Oral Digital Medicine and 3DPrinting Engineering, Qingdao University, Qingdao266003, Shandong Province,China Corresponding author:Sun Jian, M.D., Professor, Chief physician, Master’ssupervisor, Department ofOral and Maxillofacial Surgery, the Affiliated Hospital of QingdaoUniversity, Laboratory of Oral Digital Medicine and 3DPrinting Engineering,Qingdao University, Qingdao 266003, Shandong Province, China3D 仿生打印组织工程骨修复下颌骨缺损杜国庆，孙 健，李亚莉，陈立强，陈 晨，邓 楠，吴雨桐，李 莉，王志浩(青岛大学附属医院口腔颌面外科，青岛大学口腔数字医学与3D 打印工程实验室，山东省青岛市 266003)DOI:10.3969/j.issn.2095-4344.0868 ORCID: 0000-0002-0182-9203(杜国庆)文章快速阅读：文题释义：3D 仿生打印：3D 仿生打印机可调控打印仓内温度等相关参数来维持仓内细胞及生长因子的活性。

GTR修复牙槽骨缺损的beagle犬动物模型的研究华楠;庄晶【摘要】目的为研究PLGA膜在beagle犬牙槽骨中的生物降解效果,建立一种符合牙槽骨缺损特点的动物模型.方法该研究在2011年12月-2012年2月期间,以3只beagle犬为实验动物,年龄在14～16个月,平均体重(13.0±1.0)kg.拔除其双侧前磨牙.拔除牙后同时放入可吸收膜.在拔牙窝的左侧放置Bio-Guide胶原膜,右侧放置PLGA膜.结果 3只实验犬均无死亡,饮食、活动正常.手术4周后再次进入牙槽骨,发现胶原膜和PLGA膜被完全吸收,生物降解.光学显微镜下显示在放置胶原膜和PLGA膜的拔牙窝上都形成了新的骨小梁.结论该研究采用的建模方法,成功构建了与临床即刻种植骨缺损相似,并可作为研究即刻种植骨界面骨性结合的实验动物模型.【期刊名称】《中外医疗》【年(卷),期】2015(034)026【总页数】2页(P48-49)【关键词】牙槽;骨缺损;引导;组织再生;动物实验模型【作者】华楠;庄晶【作者单位】上海杨思医院口腔科,上海200126;上海杨思医院口腔科,上海200126【正文语种】中文【中图分类】R780.1引导骨再生技术（GBR）是针对骨的引导组织再生（GTR）技术，由GTR延伸而来[1]，GTR主要应用于牙齿[2]。

目前，GBR主要运用的是胶原蛋白膜，然而它具有局限性，因此有必要研究合成聚酯纤维的可吸收生物膜。

聚乳酸-羟基乙酸（PLGA）膜是一种可吸收生物膜，具有很好的生物降解性和生物相容性。

而建立动物模型，研究PLGA膜在动物牙槽骨中的生物降解效果，才能进一步阐明PLGA 作为GBR膜在临床使用中的效果。

多位学者先后采用猴、小型猪、比格犬、大耳白兔、鼠等多种实验动物，在其下颌骨制造骨缺损，植入植骨材料，采用组织学的方法评估植骨材料[3]。

但其动物实验模型与种植临床的骨缺损形态各不相同。

为研究PLGA膜在beagle犬牙槽骨中的生物降解效果，建立一种符合牙槽骨缺损特点的动物模型，该研究在2011年12月—2012年2月，采用beagle犬，年龄在14～16个月，按照天然骨缺损动物模型建立的方法制备牙槽骨缺损的实验模型[4]，现报道如下。

壳聚糖加强骨组织生物修复机理解析骨组织是人体中最重要的结构之一，具有支撑身体、保护内脏和参与骨骼运动等重要功能。

然而，骨组织容易受到外界损伤和疾病的侵袭，导致骨折、骨质疏松和骨缺损等各种问题。

为了促进骨组织的愈合和修复，许多研究人员致力于寻找新的治疗方法和材料。

壳聚糖作为一种生物可降解的聚合物材料，已被广泛研究，其在骨组织生物修复中具有重要的作用。

壳聚糖是一种天然聚合物，主要存在于海洋生物中，如虾、蟹、贝壳等。

它具有生物相容性、生物降解性和生物可吸收性等优良特性，对人体无毒副作用，因此被广泛应用于医学领域。

壳聚糖加强骨组织生物修复的机理主要涉及以下几个方面：1. 促进骨细胞生长和增殖：研究表明，壳聚糖能够促进骨细胞的增殖和分化，从而提高骨组织的新生和修复。

壳聚糖可以通过调控一系列与骨细胞增殖和分化相关的信号通路来促进骨细胞的再生。

例如，壳聚糖可以通过激活Wnt/β-catenin信号通路和增加TGF-β1的分泌来促进骨细胞增殖和骨基质合成。

2. 促进骨基质沉积：壳聚糖可以促进骨细胞合成和分泌骨基质，从而增加新骨的沉积，并加强骨组织的力学性能。

壳聚糖可以通过刺激骨细胞产生胞外基质蛋白、胶原蛋白和骨基质硫酸葡聚糖等物质，促进骨基质的形成和沉积。

3. 促进血管生成：血管生成是骨组织修复过程中必不可少的一步，壳聚糖通过促进血管内皮细胞的增殖和迁移，以及诱导血管生成因子的表达，可以增加新生血管的密度和分布，提供充足的血液供应和氧气供应，促进骨组织的修复和再生。

4. 抗炎作用：壳聚糖具有抑制炎症反应的作用，可以通过抑制炎性介质的释放和炎症信号通路的调节，减轻骨组织受损区域的炎症反应，促进骨组织的修复和再生。

5. 促进骨组织再生的免疫调节作用：壳聚糖可以调节免疫反应，并促进骨组织再生。

壳聚糖可以通过调节T细胞的分化和功能、调节巨噬细胞的活化和极化等机制，促进骨组织的修复和再生。

总之，壳聚糖作为一种生物可降解的材料，在骨组织生物修复中具有重要的作用。