Food Chemistry-Novel molecular imprinted polymers over magnetic mesoporous silica

专利名称：一种马铃薯腐烂茎线虫脂肪酸去饱和酶基因序列专利类型：发明专利
发明人：魏利辉,邵颖,万景旺,赵雷,张红艳
申请号：CN201210075653.7
申请日：20120321
公开号：CN102634526A
公开日：
20120815
专利内容由知识产权出版社提供
摘要：本发明公开了一种马铃薯腐烂茎线虫脂肪酸去饱和酶基因序列，该基因cDNA全长
1183bp，自110bp至1108bp区段为其开放阅读框，编码333个氨基酸，5’非编码区长
109bp，3’非编码区长75bp，有多聚核苷酸尾巴，该基因在线虫耐寒作用机制中起作用，同时还在抗线虫转基因作物方面应用。

申请人：江苏省农业科学院
地址：210014 江苏省南京市玄武区钟灵街50号
国籍：CN
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菜豆多聚泛肽基因在重金属胁迫下的表达_英文_ () 植物学报 1999 , 41 10:1052,1057Acta B ota nica Si nicaΞ菜豆多聚泛肽基因在重金属胁迫下的表达柴团耀张玉秀( )中国科学技术大学研究生院生物部北京 100039( ) 摘要差别筛选 HgCl胁迫的菜豆 Phaseolus vulgaris L . 幼苗叶片 cDNA 库 , 分离出两个重金属胁迫相应基因 2 ( ) PvS R5 和 PvS R51 Phaseolus vulgaris stress- related gene片段。

cDNA 和氨基酸序列分析表明 PvS R5 和 PvS R51 分别编码一种多聚泛肽。

Northern blot 分析表明多聚泛肽是组成性表达蛋白 ,主要在根中表达 ,叶片和茎中表达较少 ; Hg 、Cd 、Cu 和 Zn 等重金属、高温和水杨酸能强烈地刺激其在叶片中的表达 ,而受伤几乎没有影响。

推测多聚泛肽在抵抗重金属胁迫和提高植物的抗逆性方面有重要作用。

关键词重金属 ,多聚泛肽 ,菜豆Expression Analysis of Polyubiquitin Genes from BeanΞ in Response to Heavy MetalsCHAI Tuan- Yao ZHANG Yu- Xiu( )Department of Biology , Graduate School of University of Science and Technology of China , The Chinese Academy of Sciences , Beijing 100039( Abstract Using differential screening of a leaf cDNA library prepared from a bean cultivar Phaseolus vul2) garis L . cv. Saxaexposed to HgCl, the authors have isolated and characterized two heavy metal- regulated 2( ) cDNA fragments , designated as PvS R5 and PvS R51 Phaseolus vulgaris stress- related gene. The sequencesof the cDNA inserts and homological analysis showed that both PvS R5 and PvS R51 encode a polyubiquitin re2spectively. The polyubiquitin genes were constitutively expressed in roots but weakly expressed in stems andleaves. Northern blot analysis revealed a low level of transcripts of polyubiquitin in unstressed bean leaves , butthe gene expression was strongly stimulated by heavy metals , elevated temperature and salicylic acid , whereaswounding had almost no effect . These suggested that polyubiquitin might play important roles in resistance toheavy metals and various environmental stresses.Key words Heavy metals ,Polyubiquitin , Phaseolus vulgarisUbiquitin is a 76 amino acid protein highly con2 mal protein , the ubiquitin- dependent pathway controls the served in all eukaryotes. The ubiquitins are encoded by levels of many key cell regulators , i . e . phytochrome andcyclin. So it plays major roles in various biological pro2 two different gene families , polyubiquitin genes and ubiq2 1 ,2 ) ( uitinextension protein genes UbEP . Polyubiquitin cesses, including DNArepair , transcription , signaltransduction , the cell cycle control , apoptosis and stress genes contain several direct repeats of the ubiquitin codingunit . UbEP genes contain a single ubiquitin coding unit responses. The expression of polyubiquitin genes can be 3 ,4 fused in frame to the coding region for a small protein as2 enhanced by HgClin maize and tobacco , we report 2sociated with ribosomes. Ubiquitin monomer in the cyto2 here the identification and characterization of polyubiqui2 plasm can be covalently attached to other proteins via a tin cDNA from bean and gene expression in response toheavy metals. Furthermore , the mechanism of plant resis2 multistep enzymatic process. The linkage occurs at specif2tance to heavy metals is discussed. ic lysine residues of the acceptor protein with formation ofan isopeptide bond between the carboxyl group of the C- 1 Materials and Methods εterminal glycine of ubiquitin and- NHgroup s of the ly2 2 sine side- chains of the target protein. The conjugation of 111Plant materials and stress conditions ubiquitin to protein may alter their stability or serve as a ( ) Bean Phaseolus vulgaris L . cv. Saxaseeds were recognition signal for proteolysis by the 26 S proteo2 surface- sterilized with a 2 . 5 % calcium hypochloride solu2 1 some .In addition to removing the denatured or abnor2 tion for 10 min , rinsed several times with distilled water() Ξ Supported by the“863”Grant for Youth and the NationalNatural Science Foundation of China No . 39870078. Received : 1999- 01- 14 Revised : 1999- 05- 16and then imbibed in sterile water for 16 h. Germination signal with the control probe . The two clones were sub2 ?after which the young plants were trans2 occurred at 22 ( ) cloned into pBluescript KS + plasmid vector and se2 ferred onto a liquid culture medium containing KNO2 3 quenced by the dideoxy method.( ) mmol/ L , Ca NO2 . 5 mmol/ L , MgSO1 mmol/ L , 3 2 4 113Northern blot analysisKHPO1 mmol/ L and Fe 2 . 8 mg/ L , Mn 0 . 55 mg/ L , 2 4 RNA samples were prepared and analyzed according 6 Zn 0 . 65 mg/ L , Cu 0 . 06 mg/L , B 0 . 32 mg/ L , Mo 0 . to standard protocols, by electrophoresis on 1 . 2 % a2 02 mg/ L . Plants were grown in a growth chamber with a garose-formaldehyde gels , transferring onto Hybond N photoperiod of 16 h at 22 ? during the day and 18 ? membranes , and hybridizing for 24 hin the presence of 6 during the night and a photosynthetic photon flux density ( ×SSC 1 ×SSC : NaCl 0 . 15 mol/ L , CHNaO0 . 15 6 537 32 - 2 - 1 ) μmol/ L- 50 % formamide at 42 ? to P- cDNA probes. When the two primary leaves of 150 mol m? s? .Hybridization was followed by three washes for 15 min were well expanded , plants were stressed by adding one ofeach at 42 ?in 2 ×SSC. Finally , the membrane was ex2the following metals : HgCl, CdCl, CuSOor ZnSOat a 2 2 4 4μposed to X- ray film for autoradiography at - 80 ?.final concentration of 100mol/ L . For other experiments() plants were grown in plots in soilunder the same light- 2 Results dark and temperature conditions. Various types of abiotic PvSR51 encode a polyubiquitin re2 2 . 1 PvSR5 and stress were applied asfollows : leaves were sprayed with a spectively ( ) 0 . 2 % W/ VHgClor CdCl, or 10 mmol/ L salicylic 2 2 analysis showed that PvS R5 clone con2 Sequence ( ) acid SA solution. For heat- shock treatment , plants ( ) tained a 790 bp insert Fig. 1A. Northern blot revealed were placed at42 ? for 4 h , while wounding was per2 7 that PvS R5 mRNA has a size of 1 200 nucleotides, in2 formed by dusting the leaves with celite and gentle rub2 dicating that the cDNA was not full- length. PvS R5 cDNA bing. Leaf tissue was harvested at various time points after contains more than two repeats of ubiquitin coding unit stress treatment . and lacks the 5′end coding sequence , leucine which is 2 bean cD NA li12 Construction and screening of a 1unique non- ubquitin residue at the end of the last ubiqui2 brary tin repeat . Total RNA extraction was done using the phenol/ PvS R51 cDNA is also partial fragment that lacks the 5 chloroform/ isoamylalcohol technique. Polyadenylated 5′coding region.It encodes more than four ubiquitin re2mRNAs were obtained by chromatographing total RNA peats , the terminal repeat contains another additional ( ) twice on oligo- dTcellulose as described by Sambrook ( )amino acid , phenylalanine Fig. 1B. Repeats of 228 nu2 6 et al . Double- stranded cDNA was synthesized from cleotides encoding ubiquitin monomers were aligned below polyadenylated RNA isolated from bean plantsharvested 6 the first complete one taken as an arbitrary reference . Ho2 h after spraying with mercuric choloride solution using the mologies were found to range between 80 % to 90 %. Pharmacia Biotech kit . The cDNA library was constructed There are no homologies in the3′untranslated region be2 λin the bacteriophagegt 10 cloning vector . tween PvS R5 and PvS R51 genes. Therefore , PvS R51 en2 The cDNA clones were placed at a density of about codes a polyubiquitin that isdifferent from PvS R5 . ( ) 1 000 plaques/ dish diameter 9 cm. Duplicate sets of 2 . 2 Expression of PvSR5 gene in various tissues of nitrocellulose filters containing recombinant phage plaques young bean plants 32 were screened with [ P - labelled cDNA probes , pre2 PvS R5 gene is highly expressed in root but weakly pared of using polyadenylated RNAs extracted from mer2 ( ) expressed in stems and leaf tissue Fig. 2. The same curic chloride- treated or untreated bean leaves. Differen2 ( pattern was observed on PvS R51 gene results not tial screening permitted the isolation of clones that were ) shown, demonstrating that polyubiquitin genes are con2 only expressed in mercury- treated plants or that were ex2 stitutively expressed in different tissues. pressed at higher levels in stressed plants than in control PvS R5 gene expres2 Effect of heavy metals on 2 . 3 plants. Both types of clones were considered to be heavy- sion in primary leaves( metal stress- related. 32 positive clones Phaseolus vul2 Theexpression of polyubiquitin gene was studied in ) garis stress- related gene , PvS R were isolated by screen2 response to several metal stresses. More than one hy2 ing of the cDNA library , in which PvS R5 and PvS R51 bridization signals are observed in Fig. 3 , as PvS R5 clones gave a strong hybridization signal with the cDNA probe contains partialubiquitin coding sequence that is probe obtained from mercury- treated plants and a weak植物学报 1054 41 卷() ()Fig. 1 Nucleotide sequence and deduced amino acid sequence of PvS R5 cDNA Aand PvS R51 cDNA B A. The initiation amino acid of the ubiquitin repeats is boxed , asterisk indicates the stop codon. GenBank access number for PvS R5 is U77940. B. The stop codon is indicated by an asterisk and the putative polyadenylation signal is underlined.Nucleotide sequences of the ubiq2 uitin repeats are aligned. Identical nucleotides are indicated by dots. Amino acid sequences are given in the one- letter code below the last u2 biquitin repeat . GenBank access number for PvS R51 is U77939.levels in the leaves , but with a different kinetics of induc2tion from that obtained with sprayed leaves. When mer2curic chloride was absorbed by the roots , PvS R5 mRNAstarted to accumulate at 9 h after the onset of treatment ,( ) reached a maximum at 48 h and then declined Fig. 3C.The transcript level continued to increase from 9 h afterthe onset of treatment until a maximum was reached 48 h( ) later when cadmium was absorbed by the roots Fig. 3D. Root- absorbed copper also stimulated the PvS R5 expres2 Fig. 2 PvS R5 gene expression in various tissues of Phaseolus vul2 garis seedlings sion , the transcripts reached a maximum after 24 h , ( μ) Total RNAs 10 g/ laneextracted from the various tissues of 12( ) whereas zinc had alittle effect Fig. 3 E , F. day-old plants were separated on formaldehyde- agarose gels , trans2 2 . 4 PvSR5 gene regulation by otherf orms of a biotic ferred onto membranes and hybridized with PvS R5 cDNA. R. Root ;S. Stem ; L . Primary leaves. stress Spraying with SA resulted in a large increase in tran2 highly homologous to the other ubiquitin gene . As shown script levels , reached a maximum at 3 h and then de2 in Fig.3A and 3B , the amounts of PvS R5 transcripts in2 ) ( ) (clined Fig. 4A. High temperature 42 ?also stimu2 creased rapidly , reached a maximum at3 h and then de2 ( ) lated strongly PvS R5 gene expression Fig. 4B, PvSR5 creased gradually when the leaves were sprayed with mer2 ( mRNA reached a maximum at 1 h during the stress 4 curic chloride or cadmium chloride solution. When the ) h, and then decreased after plants were returned to nor2 mercuric chloride or cadmium chloride was added to the mal temperature condition. Wounding had almost no effect liquid culture medium instead of being sprayed onto the ( ) PvS R5 gene expression Fig. 4C. on plants , PvS R5 mRNA also accumulated above the basalFig. 3 Northern blots analysis showing the levels of PvS R5 mRNA in bean leaves treated with various metals () () The time course of accumulation of PvS R5 transcripts after the seedlings were treated byvaporization of HgClAand CdClB, or by root- 2 2 ( ) () ( ) ( ) absorption of HgClC, CdClD, CuSOE, and ZnSOF. 2 2 4 4encode a polyubiquitin respectively. Both PvS R5 and 3 Discussion PvS R51 cDNA are incomplete at their 5′end probablySequence analysis showed that PvS R5 and PvS R51 because of inverted repeats in coding regions of ubiquitin genes contained more than two repeats of the ubiquitin mRNAs forming snap back loop structures which are self- 4 coding unit . The additional amino acid residues of both primed during cDNA synthesis. PvS R5 mRNA prefer2 genes encoded are different , PvS R5 terminates at entially accumulated in the root of bean seedling indicat2 leucine , whereas the phenylalanine is found at the C- ter2 ing that polyubiquitin gene has tissue- specific expression. minal of PvS R5 , suggesting that PvS R5 and PvS R51植物学报 1056 41 卷stimulate the expression of PvS R5 , but Cu and Zn had a( ) little effect Fig. 3, demonstrating that Hg and Cd ions are more toxic than Cu and Zn to living cell .PvS R5 gene responds not only to heavy metals , butalso to other stresses such as elevated temperature , 13 12 and virus infection, indicating that the ubiqui2 UVtin pathway has important roles in eliminating the damagedproteins induced by various stresses and maintaining thecell structure and function. Transgenic rice experimentsshowed that polyubiquitin gene expression was limited tothe region exposed to heat stress and/ or wounding , oraround the necrotic lesion induced by pathogen infection , 14 but rather not regulated systemically. This implied thatpolyubiquitins have key roles in the processes of the localdefence reaction. SA is a natural signal in the inductionof defense responses , which accumulates upon ozone or 15 Fig. 4 Northern blot analysis showing the time course of PvS R5 UV treatment , as well as pathogen attack. Exogenous accumulation in bean leaves under various abiotic stresses SA can positively regulate the gene expression of PvS R5 , The arrow on panel B indicates the transfer of the stressed plants to leading to the conclusion that SA might be involved in the normal growth conditions and the time points behind the arrows the duration of recovery. A. Salicylic acid ; B. Heat stress ; C. pathway of polyubiquitin mRNA synthesis in response to Wounding. 16 stresses. Chen et al . proposed that systemically ac2quired resistance signaling is mediated by an accumulation PvS R5 transcripts was detected in un2 A low level ofof HO, because SA specifically binds to catalase and ( ) stressed bean leaves Fig. 3, but the gene expression was 22 2 + 2 + inhibits its activity , leading to an elevation of HOlevel . ( 22 strongly stimulated by heavy metals Hg, Cdand 2 + Furthermore , HOinduces expression of defense- related 22 ) Cu. The datum is consistent with the responses of genes. A rapid and transient release of active oxygen polyubiquitin genes to mercuric stress in maize and tobac2 9 3 ,4 () species AOScan be induced by heavy metals, UV , , suggesting that polyubiquitin may playan impor2 co 16 17 pathogen infection, SA and heat stress. These re2tant role in resistance to heavy metals. Heavy metal ions sults suggest that PvS R5 gene expression was possibly in2 can bind to protein sulfhydryl group s , leading to deficien2volved in the regulation of AOS in response to stresses , cy of essential ions and destruction of the enzyme struc2 8 ,9 and there may be a common signal transduction pathway ture . Other toxicity mode is oxidative damage by free 9 ,10for various stresses in the regulation of PvS R5 gene ex2 radicals generated by metal redox cycling . Eventual2 pression. ly , the two processes result in accumulating huge amount of denatured and damaged proteins by metals in the cell .References In eukaryotes , an important selective proteolysis pathway for the elimination of abnormal proteins that are generated Belknap W R , Garbarino J E. The role of ubiquitin in plant 1 senescence and stress responses. Trends Plant Sci , 1996 , 1 : under normal or stress conditions is ATP- dependent and 2 331,335 mediated by ubiquitin system. The ubiquitin monomers generated by UbEP genes act as a molecular“chaperone” Tanaka K. Proteasones : Structure and biology. J Biochem , 21998 , 123 : 195,204in facilitating ribosome assembly. In contrast , the polyu2 Didierjean L , Frendo P , Nasser W , Geneviève G , Marivet J , 3 biquitin genes would provide monomers for general utiliza2 Burkard G. Heavy metal- responsive genes in maize : Identifi2 1 ,2 tion inubiquitin- mediated processes in cell. In cation and comparison of theirexpression upon various forms of yeast , the expressions ofpolyubiquitin and ubiquitin- con2 abiotic stress. Planta , 1996 , 199 : 1,8jugating enzyme genes are activated in response to cadmi2 4 Genschik P , Parmentier Y , Durr A , Marbach J , Criqui M C.um exposure and the mutants deficient in specific ubiqui2 Ubiquitin genes are differentially expressed in protoplast- de2rived cultures of Nicotiana sylvestris and in response to varioustin- conjugating enzymes are hypersensitive to cadmium , stresses. Plant Mol Biol , 1992 , 20 : 897,910 proposed that cadmium resistance is meditated partially by 11 Ragueh F , Lescure N , Roby D , Marco Y. Gene expression in 5 ubiquitin pathway. PvS R5 mRNA levels are increased Nicotiana tabacum in response to compatible and incompatible upon treatment with metals , implying that enormous ubiq2 isolates of Pseudomonas solanacearum . Physiol Mol Plant uitin monomers are required for degrading the abnormal Pathol , 1989 , 35 : 23,33 proteins produced by metals. Hg and Cd can strongly 6 Sambrook J , Fritsch E F , Maniatis T. Molecular Cloning : A() ()Laboratory Manual . 2nd ed. New York : Cold Spring Harbor 生物物理学报,1998 , 14 : 767,771 in Chinese() ( ) 13 Zhang Y- X张玉秀, Chai T- Y柴团耀. Effects of alfalfa Laboratory Press , 1989.) ( ) ( 7 柴团耀 , Zhang Y- X 张玉秀 , Burkard G. Chai T- Y mosaic virus infection on the expression of stress- responsiveHeavy metal- responsive genes in bean : cloning of cDNAs and ()genes in bean. Acta Hort Sin 园艺学报,1998 , 25 : 399,(()gene expression analysis. Acta Phytophysiol Sin 植物生理学 401 in Chinese ) ()14 Takimoto I , Christensen A H , Quail P H , Uchimiya H , Toki 报, 1998 , 24 : 399,404 in ChineseS. Non- systemic expression of a stress- responsive maize polyu2 8 Van Assche F , Clijsters H. Effects of metal on enzyme activity in plants. Plant Cell Environ , 1990 , 13 : 195,206 () biquitin gene Ubi-1in transgenic rice plants. Plant Mol Bi2 ( ) ( ) Zhang Y- X 张玉秀 , Chai T- Y 柴团耀 , Burkard G. ol , 1994 , 26 : 1007,1012 9 (Heavymetal tolerance mechanisms in plants. Acta B ot Sin 植 Ec Key- Kaltenbach H , Kiefer E , Grosskopf E , Ernst D , San2 15 ) ()物学报, 1999 , 41 : 453,457 in Chinese dermann H. Differential transcript induction of parsley patho2 González A , Steffen K L , Lynch J P. Light and excess man2genesis- related proteins and of a small heat shockprotein by o2 10 ganese implications for oxidative stress in common bean. Plant zone and heat shock. Plant Mol Biol , 1997 , 33 : 343,350 Physiol , 1998 , 118 : 493,50816 Chen Z , Silva H , Klessig D F. Active oxygen species in the J ungmann J , Reins H A , Schobert C , J entsch S. Resistance to 11 induction of plant systemic acquired resistance by salicylic cadmium mediated by ubiquitin- dependent proteolysis. N a2 acid. Science , 1993 , 262 : 1883,1886 ture , 1993 , 361 : 369,371 17Dat J F , Lopez- Delgado H , Foyer C H , Scott I M. Parallel () () (Chai T- Y柴团耀, Zhang Y- X张玉秀, Zhang Z- D 张正 changes in HOand catalaseduring thermotolerance induced 12 22 ) 东. Effects of elevated ultraviolet radiation on the stress- re2 by salicylic acid or heat acclimation in mustard seedlings. sponsive gene expression of bean seedling. Acta Biophys Sin Plant Physiol , 1998 , 116 : 1351,1357。

蜂毒素抑制博莱霉素诱导小鼠肺纤维化的机制研究李莉危蕾王众福张秀莲钱叶长（1上海中医药大学附属曙光医院宝山分院，上海，201900；2上海市宝山区中西医结合医院，上海，201900）摘要目的：观察蜂毒素对博莱霉素诱导小鼠肺纤维化的干预作用。

方法：70只SPF级C57BL/6小鼠被随机分为正常组10只和模型组、地塞米松组、蜂毒素低剂量组、蜂毒素中剂量组、蜂毒素高剂量组各12只。

除正常组外，均采用气管穿刺注入博莱霉素（BLM）制备肺纤维化小鼠模型。

从术后第1天开始，地塞米松组按3mg/kg的剂量腹腔注射，蜂毒素低、中、高剂量组分别给予5μg/（kg·d）、10μg/（kg·d）、20μg/（kg·d），对照组和模型组给予等体积生理盐水灌胃，连续2周。

分别于第7、第14天处死动物，收集小鼠外周血样本，通过ELISA方法检测血清转化生长因子（TGF-β1）、胶原蛋白I （CollagenI）、胶原蛋白III（CollagenIII）、基质金属蛋白酶2（MMP2）和基质金属蛋白酶9（MMP9）的水平。

取肺组织进行苏木精-伊红（HE）分析，Masson染色和羟脯氨酸（HYP）评估以观察组织病理学变化和胶原沉积。

采用实时荧光定量（Ｒeal-ime PCＲ）法和蛋白兔疫印迹法（Western blot）观察各组大鼠肺组织TGF-β1、Smad2、Smad3等蛋白和基因的表达变化。

结果：与对照组比较，模型组大鼠肺纤维化明显，HYP、TGF-β1、CollagenI、CollagenI的含量升高（P＜0.05），肺组织TGF-β1、Smad2、Smad3蛋白和基因表达升高（P＜0.05）；与模型组比较，蜂毒素中高剂量组血清HYP、TGF-β1、CollagenI、CollagenI 的含量下降（P＜0.05），肺组织TGF-β1、Smad2、Smad3蛋白和基因表达降低，差异有统计学意义（P＜0.05）。

聚乙二醇改性水溶性壳聚糖对蛋白药物的释放作用王春1，扶雄2，杨连生2（1.广东石油化工学院化学与生命科学学院，广东茂名 525000）（2.华南理工大学轻工与食品学院，广东广州 510640）摘要：本文采用离子交联的方法，研究制备了聚乙二醇（PEG）修饰的水溶性壳聚糖（WSC）药物载体。

以牛血清蛋白（BSA）作为模型蛋白药物，对纳米粒子的物理化学性质作了初步检测。

由于分子间的竞争作用，PEG的修饰在一定程度上降低了WSC纳米粒子的药物负载能力。

体外释放实验表明PEG修饰的纳米粒子在一定程度上加快了BSA的释放，但仍具有较好的缓释性能。

关键词：水溶性壳聚糖；聚乙二醇；纳米粒子；牛血清蛋白；蛋白质释放文章篇号：1673-9078(2011)8-885-886PEG Modified Water-soluble Chitosan Nanoparticles for Drug DeliveryW ANG Chun1, FU Xiong2, YANG Lian-sheng2(1.Chemistry and Life Science College, Guangdong University of Petrochemical Technology, Maoming 525000, China)(2.College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China)Abstract: PEG modified WSC nanoparticles were prepared based on ionic gelation and the capability of PEG modified water-soluble chitosan (WSC) was used as carriers to load and delivery drug. As a model protein drug, bovine serum albumin (BSA) was incorporated into the nanoparticles. Physicochemical of characterizations of WSC nanoparticles was determined preliminarily. PEG introduction might decrease loading capability of nanoparticles due to the competition effect between BSA and PEG. And in vitro release demonstrated that PEG modification could increase release of loading protein drug but the nanoparticles displayed a good release performance still.Key words: water-soluble chitosan (WSC); PEG; nanoparticle; BSA; protein delivery聚乙二醇(PEG)是一种被广泛应用于生物医用材料的高分子，它和水溶性壳聚糖（WSC）一样具有生物相容性好、无毒无副作用、并可生物降解等特点。

收稿日期:20052032301基金项目:甘肃省自然科学基金项目(ZR 2972068)1硒酸酯多糖的免疫调节与抗肿瘤作用张哲文1,魏虎来2,苏海翔2(11兰州大学基础医学院;21兰州大学医学实验中心,甘肃兰州　730000) 硒(Se )是人类必需的微量元素之一,多年来国内外对硒的生物学功能进行了大量研究,尤其是硒与肿瘤的关系,已成为硒生物学功能研究中最为令人关注的领域之一,并取得了重大进展[1]。

流行病学调查、动物实验和临床应用研究证实硒是许多肿瘤强有力的预防剂、抑制剂和治疗辅助剂,能够减轻化疗药物的毒副作用,增加机体对抗肿瘤药物的耐受性而不影响其抗癌活性。

近年的研究表明,与亚硒酸钠等无机硒相比,硒酸酯多糖、硒酵母、富硒麦芽等有机硒化合物具有生物活性高,硒的生物利用度高,不易蓄积,毒性极低等特点,更适合于人体生理和临床应用。

随着生命科学的发展,多糖和硒元素在生命过程中的重要作用日渐引人注目,本文重点综述一种全新的有机硒化合物硒酸酯多糖的免疫调节作用和抗肿瘤效应。

1　硒酸酯多糖的生物特性卡拉胶是从红藻中提取的天然植物胶。

分为Kappa 型(由鹿角藻中提取)和Iota 型(由麒麟菜中提取),其化学本质为硫酸酯多糖。

硒酸酯多糖又称硒化卡拉胶(Kappa 2Selenocarrageenan ,KSC ),是人工制备的卡拉胶的硒化产物。

卡拉胶是β2D 2吡喃半乳糖与3,62内醚222D 2吡喃半乳糖以C1,3键和C1,4键交替连接构成的无分支链型硫酸酯多糖。

其结构中的硫部分地被硒取代,即形成硒酸酯多糖。

此有机结构能够有效地提高硒的生物利用度和生理功能,且毒副作用比无机硒大大降低,是一种安全、有效、健康的富硒化合物[2]。

KSC 具有硒和多糖的双重功效,具有优势互补,彼此协同增效的特点,具有广泛的生理活性,兼具有机硒和多糖的诸多生理功能,已证明KSC 在体内外具有显著的抗肿瘤免疫调节作用,可抑制肿瘤细胞增殖、增强免疫功能和降低化疗药物的毒副作用等。

Vol．31高等学校化学学报No．112010年11月CHEMICAL JOURNAL OF CHINESE UNIVERSITIES 2184 2189生物素修饰纳米银探针的制备及在蛋白芯片可视化检测中的应用李慧1，钟文英1，许丹科2（1．中国药科大学基础部分析化学教研室，南京210009；2．南京大学化学化工学院，南京210093）摘要采用寡核苷酸为连接分子成功制备了生物素修饰的纳米银探针，并建立了纳米银催化同种金属离子的特异性还原显色反应．实验采用蛋白质芯片为分析工具，以微量人IgG 为蛋白分析模式研究了纳米银探针/氢醌/硝酸银体系的显色分析性能．实验结果表明，上述检测体系可对160fg 100pg 含量范围内的微量蛋白显示可视化结果，蛋白点的灰度值与其浓度具有良好的相关性，最小蛋白检测量可达160fg．同时还开展了与商品化链亲和素纳米金/银增强试剂显色方法的对比实验，结果表明，本法制备的探针对蛋白的检出限降低了约40倍，且具有存储稳定、反应快速等优点．关键词生物素修饰纳米银；氢醌/硝酸银；微量蛋白检测；蛋白芯片中图分类号O657．39文献标识码A 文章编号0251-0790（2010）11-2184-06收稿日期：2010-03-12．基金项目：国家“九七三”计划项目（批准号：2006CB910803）和蛋白质组学国家重点实验室开放课题资助．联系人简介：钟文英，女，博士，副教授，主要从事量子点的合成及分析应用和蛋白芯片检测新技术研究．E-mail ：wyzhong@cpu．edu．cn许丹科，男，博士，教授，主要从事生物阵列传感器件及蛋白质芯片领域的研究．E-mail ：xudanke@nju．edu．cn 随着纳米材料科学的发展，研究具有检测生物分子独特功能的纳米试剂已成为纳米材料研究的前沿．其中纳米金已经被广泛应用于免疫分析［1］、免疫层析［2］及显色反应等领域．此外，纳米金也被进一步用于催化银增强显色反应研究［3 7］．近年来，具有优良光学特性的纳米银正被越来越多地应用于蛋白质［8］和DNA ［9 11］等生物分子的检测分析［12，13］．纳米银具有摩尔消光系数高、表面增强拉曼散色效应强和催化活性好等独特的物理化学性能［8］，但其良好的催化显色特性并未见应用于微量蛋白的检测．文献［14］报道，在金属纳米颗粒上催化还原同种金属离子具有较好的灵敏度，如在纳米金上采用金增强试剂比采用银增强试剂检测蛋白质的灵敏度更高．基于此，本文开展了纳米银表面催化还原银离子的蛋白显色检测方法的研究．通过以蛋白芯片分析为模式进一步研究了纳米银探针高灵敏、快速检测微量蛋白的可能性．微量蛋白通过微阵列点样仪固定于醛基修饰的载玻片表面，依次加入的生物素标记的羊抗人IgG 、亲和素以及生物素修饰纳米银探针/氢醌/硝酸银检测试剂可与微量蛋白发生特异性的显色反应．实验对可能影响可视化检测效果的探针和氢醌/硝酸银浓度以及相关反应条件进行了优化，并将所建立的方法与商品化的纳米金/银增强显色试剂的分析结果进行了系统比较，对此方法的优势进行了探讨．1实验部分1．1材料、试剂与仪器醛基修饰的载玻片（上海百傲科技有限公司），生物素标记的羊抗人IgG （Bio-gahIgG ，美国KPL 公司），亲和素标记的胶体金（SA-AuNPs ）和银增强试剂A ，B （美国Sigma 公司），寡核苷酸PA ［5'SH-（CH ）6-AAAAAAAAAAAAAAA3'-Biotin ］（上海生工生物工程公司）；牛血清白蛋白（BSA ）、人IgG 及1ˑPBS （137mmol /L NaCl +2.7mmol /L KCl +10mmol /L +Na 2HPO 4·12H 2O +2mmol /L KH 2PO 4）均购自南京布克生物有限公司，氢醌（分析纯，南京化学试剂有限公司），硝酸银（分析纯，上海申博化工有限公司），硼氢化钠（分析纯，天津市化学试剂研究所），吐温-20（天津市科密欧化学试剂开发中心）．微阵列点样仪（博奥生物技术有限公司），Scanmaker i900型扫描仪，LuxScan3．0芯片图像分析软件（北京博奥生物技术有限公司），JEM-2100型透射电子显微镜（日本JEOL 公司）．1．2实验步骤1．2．1银纳米粒子的制备参照文献［15］的方法，在冰浴条件下，将40mL 2mmol /L 的硝酸银溶液逐滴加入至80mL 3mmol /L 硼氢化钠溶液中，不断搅拌至反应完全，继续搅拌至室温，得到纳米银溶液，常温保存．1．2．2生物素修饰银纳米粒子探针的制备参照文献［16］的方法，将1mL 纳米银溶液与10μmol /L 寡核苷酸PA 混合放置18h ，加入122μL 1ˑPBS ，静置6h 后，加入2mol /L NaCl 22μL ，然后每隔2h 加入2mol /L NaCl 21μL 至NaCl 的终浓度为0.1mol /L．放置48h 后，取600μL 液体离心3次（15min /次，转速为15000r /min ），沉淀用200μL 0.1mol /L PBS （0.1mol /L NaCl +0.1ˑPBS ）重悬，制得Bio-AgNPs 探针．1．2．3蛋白芯片的制备以牛血清白蛋白作为阴性对照蛋白，生物素标记的羊抗人IgG 为阳性对照蛋白，6个不同浓度的人IgG （抗原）作为样品蛋白，采用微阵列点样仪在醛基修饰的载玻片上点制18个4ˑ4蛋白点的阵列（即18个反应池，每个反应池里是一个4ˑ4的小阵列），点样量约为10nL /点；于37ħ放置2h ，4ħ放置过夜；使用前进行封闭（25μL 10mg /mL BSA ，封闭1h ）和清洗处理［1ˑPBST （1ˑPBS +0.05%Tween ）清洗2次，5min /次，氮气吹干］．1．2．4蛋白芯片的分析检测在制备好的蛋白芯片上加入5μg /mL 生物素标记的羊抗人IgG 25μL ，反应1h ，以1ˑPBST 清洗3次，加入25μL 10μg /mL SA 反应45min ，用1ˑPBST 清洗3次，加入V （Bio-AgNPs ）ʒV （0.1mol /L PBS ）=1ʒ20的25μL 检测探针反应30min ，用1ˑPBST 清洗3次，0.1mol /L PBN （0.1ˑPBS +0.1mol /L NaNO 3）清洗1次除去氯离子，干燥后加显色剂．常温下，氢醌/硝酸银反应5min ，银增强试剂反应10min．采用LuxScan3.0芯片图像分析软件采集扫描数据（文中所有的图像均用此软件处理），数据处理用灰度值进行比较．2结果与讨论与经典的酶联免疫分析反应［17］相比，基于纳米材料的催化显色反应具有试剂相对稳定及易于合成等优点．纳米金的银增强显色反应已被广泛应用于组织化学［18，19］与蛋白芯片［3 6］的图像分析中，但将其用于蛋白质检测时检出限未见明显降低．为了进一步降低检出限，本文自行设计合成了银纳米探针及显色试剂，建立了蛋白芯片上检测微量蛋白的方法，其分析流程如图1所示．Fig．1Schematic illustration of Bio-AgNPs-conjugated antibody recognition and signal amplificationwith hydroquinone /Ag +2．1纳米银探针的修饰及TEM 分析文献［9 11］报道的纳米银采用5'端修饰巯基的寡核苷酸作为探针，通过与寡核苷酸的杂交以检测目标寡核苷酸片段．本法以寡核苷酸PA 为连接分子，在寡核苷酸的5'端与3'端分别修饰巯基与生，5'端的巯基与纳米银形成稳定的配位结合，3'端的生物素特异性结合链亲和素．同时，在纳5812No．11李慧等：生物素修饰纳米银探针的制备及在蛋白芯片可视化检测中的应用Fig．2TEM image of Bio-AgNPs米银催化氢醌/硝酸银时还要利用寡核甘酸磷酸基团的负电性及其具有一定长度的特性．纳米银寡核苷酸在0.1mol /L PBS （pH =7）缓冲液中能稳定存在．图2为生物素修饰纳米银的TEM 图．从图2可见，所制备的生物素修饰的银纳米粒子的平均粒径为（17.5ʃ3.3）nm ，粒径分布为14.220.8nm．2．2氢醌和硝酸银显色剂浓度的选择氢醌和硝酸银反应的原理如下［20］：2AgNO 3+C 6H 4（OH ）→ 2CO （CHCH ）2CO +2HNO 3+2Ag ↓氢醌是常用显影剂，其浓度为1.82mmol /L．氢醌、银离子及缓冲液作为显色剂常被用于组织免疫化学［18，19，21］和蛋白芯片［3 6］研究，可被纳米金标记物特异性催化而显色．此显色剂的缺点在于，在缓冲溶液的作用下，氢醌和银离子的自身氧化还原反应会导致样品点周围的背景升高，因此选择氢醌和银离子在水溶液（pH =7）的环境下反应以减少背景信号．为考察此反应中硝酸银的合适浓度，将1mg /mL BSA 和不同浓度的链亲和素固定在醛基修饰的载玻片上，将氢醌与不同浓度的硝酸银（182，18.2和1.82mmol /L ）等体积混合后加到蛋白芯片反应池中反应．扫描检测结果显示，浓度为18.2mmol /L 的硝酸银与等体积氢醌混合后得到的信噪比最大，因此选择硝酸银浓度为18.2mmol /L ，氢醌浓度为1.82mmol /L．2．3蛋白芯片反应条件的优化2．3．1链亲和素（SA ）浓度与生物素修饰纳米银（Bio-AgNPs ）浓度的优化固定1mg /mL BSA 和不同浓度的生物素标记的羊抗人IgG ，固定抗体的浓度分别为32，160和800ng /mL 以及4，20，100和500μg /mL ，依次加入10μg /mL SA 和Bio-AgNPs 探针［V （Bio-AgNPs ）ʒV （0.1mol /L PBS ）=1ʒ20］，其余反应条件参照芯片分析过程．扫描结果显示，固定抗体浓度低于800ng /mL 的抗体点没有信号，随着抗体浓度的增加，其灰度值随之增加（芯片扫描图见图3）．Fig．3Schematic illustration （A ）and actual images （B ）of selecting concentration of SA and Bio-AgNPs10μg /mL SA ，V （Bio-AgNPs ）ʒV （PBS ）=1ʒ20．a ．1mg /mL BSA ；b ．32ng /mL Bio-gah-IgG ；c ．160ng /mL Bio-gah-IgG ；d ．800ng /mLBio-gah-IgG ；e ．4μg /mL Bio-gah-IgG ；f ．20μg /mL Bio-gah-IgG ；g ．100μg /mL Bio-gah-IgG ；h ．500μg /mL Bio-gah-IgG．选择灰度值中等强度的抗体固定量（20μg /mL ）来优化SA 和Bio-AgNPs 的浓度，图4（图4的偏差来源于2次测量结果的平均值）说明了当抗体固定浓度为20μg /mL 时，不同SA 浓度（100，10和1μg /mL ）与不同稀释倍数的Bio-AgNPs ［V （Bio-AgNPs ）ʒV （0.1mol /L PBS ）分别为1ʒ10，1ʒ20和1ʒ40］的芯片信号变化（样品点信号值/背景值）关系．当SA 浓度为100μg /mL 时，1：10的探针产生的背景高，信噪比低，1ʒ20和1ʒ40的探针产生的信噪比高，但1ʒ20探针的偏差大；当SA 浓度为10μg /mL 时，1ʒ10，1ʒ20和1ʒ40的探针浓度产生的信噪比差别不大，其中1ʒ20信号产生的偏差最小；当SA 浓度为1μg /mL 时，信噪比降低．综合上述结果，选择10μg /mL SA 和1ʒ20（体积比）Bio-AgNPs 作为以6812高等学校化学学报Vol．31下实验的分析条件．Fig．4Selecting concentration of SA andBio-AgNPs a ．100μg /mL SA ；b ．10μg /mL SA ；c ．1μg /mL SA．V （Bio-AgNPs ）ʒV （PBS ）：Ⅰ．1ʒ10；Ⅱ．1ʒ20；Ⅲ．1ʒ40．Fig．5Selecting concentration of Bio-goat anti human IgGa ．Signal intensity ；b ．backgroud intensity．ρ（Bio-Goat anti Human IgG ）/（ng ·mL －1）：Ⅰ．50000；Ⅱ．5000；Ⅲ．500；Ⅳ．50；Ⅴ．5．2．3．2生物素标记的抗体浓度的筛选按蛋白芯片制备方法，选择人IgG 固定量为2μg /mL ，加入不同浓度的生物素标记的羊抗人IgG （50和5μg /mL 以及500，50和5ng /mL ，空白），其余反应条件参照芯片分析过程．样品点信号强度与生物素标记的羊抗人IgG 浓度的关系如图5所示（图5的偏差来源于6次测量结果的平均值）．扫描结果显示，当生物素标记的羊抗人IgG 浓度为50μg /mL 时，背景较高，图像不清晰；当生物素标记的羊抗人IgG 浓度为5μg /mL 以及500和50ng /mL 时，信号强度高，背景低，且5μg /mL 时信号最强，信号的偏差最小；当浓度为5ng /mL 时，信号明显降低．综合以上结果，选择5μg /mL 作为生物素标记的羊抗人IgG 的最佳浓度．2．4纳米银/氢醌/硝酸银试剂在蛋白芯片上的检测性能将1mg /mL BSA 和浓度为16，80和400ng /mL 以及2，10和50μg /mL 的人IgG ，50μg /mL Bio-Fig．6Detection of IgG using Bio-AgNPs a ．Linear relationship between IgG concentration and relative grayscale using hydroquinone /Ag +as color reagent ；b ．linear re-lationship between IgG concentration and relative Grayscale using silver enhancer as color reagent．gahIgG 制备成蛋白芯片，依照蛋白芯片分析方法检测、扫描，结果如图6所示．氢醌/硝酸银作为显色剂时，人IgG 浓度在16ng /mL 10μg /mL 之间，IgG 浓度与相对灰度值的对数（样品点信号值/阳性点）具有较好的相关性（如图6曲线a 所示，图6偏差均来源于6次测量结果的平均值），检出限为16ng /mL （样品点信号平均值/阳性点信号平均值约等于背景信号平均值/阳性点信号平均值+3SD ，则此样品点浓度为最低检测量），与文献［22］报道的利用芯片技术检测IgG 的方法相比，本文建立的显色法检测IgG 具有方法简单、直接且所需样品少等特点，检测浓度可低至16ng /mL ；银增强试剂作为显色剂时，IgG 浓度在80ng /mL 50μg /mL 之间具有较好的相关性（如图6曲线b 所示）．实验结果表明，氢醌/硝酸银的显色效果要优于银增强试剂，主要表现在两个方面：（1）在常温下（25ħ），前者的显色时间更短，只需5min ，而后者需要10min ，且前者产生的信号比后者强（IgG 浓度在16ng /mL 10μg /mL 范围内）；（2）前者的背景比后者低．这是由于两者反应的原理稍有不同所致，以氢醌/硝酸银为显色剂仅仅是利用纳米粒子的催化特性，当有银纳米粒子存在时，纳米粒子起催化作用可加快氢醌和硝酸银的反应，使银离子在纳米粒子表面迅速被还原而呈现显著的黑色；而没有纳米银粒子存在时，氢醌和硝酸银反应缓慢，被还原的银也较少，信号点周围的背景较低．当采用银，它被纳米粒子催化显色的同时，在缓冲溶液中其自身也较快地发生氧化还原反7812No．11李慧等：生物素修饰纳米银探针的制备及在蛋白芯片可视化检测中的应用应，使信号点周围产生黑色沉淀，即背景高．2．5与商品化链亲和素纳米金试剂的比较链亲和素修饰纳米金与银增强试剂的显色结果显示，当人IgG 浓度在625ng /mL 20μg /mL之间Fig．7Detection of IgG using SA-AuNPs a ．Linear relationship between IgG concentration and relative grayscale using hydroquinone /Ag +as color reagent ；b ．linear re-lationship between IgG concentration and relative grayscale using silver enhancer as color reagent．时，人IgG 浓度与相对灰度值的对数（样品点信号值/阳性点）具有相关性（如图7曲线b 所示，图7偏差均来源于6次测量结果的平均值）；纳米金与氢醌/硝酸银的显色结果显示，当人IgG 浓度在625ng /mL 20μg /mL 之间时与相对灰度值的对数具有相关性（如图7曲线a 所示）．实验结果表明，显色剂对纳米金探针的显色能力较弱．其主要的原因有两方面：（1）两种探针修饰结构不同，导致两种纳米粒子的催化作用不同．纳米银是由具有一定长度的寡核苷酸修饰的，显色剂可以和纳米粒子的整个表面接触，因而纳米粒子可以充分发挥其催化作用；而纳米金是利用生物大分子链亲和素修饰的，部分表面被占据，无法起到催化作用，因而催化能力较低；（2）寡核苷酸上有很多带负电的磷酸基团，能静电吸附Ag +，使Ag+快速、大量地聚集在纳米银粒子表面被催化还原而迅速显色；且在反应的过程中，1分子链亲和素可以和3分子纳米银探针反应，增加了探针的结合量．所以此种修饰的银纳米粒子的催化特性更优良．为了对纳米金和纳米银探针及其两种显色剂进行比较分析，上述实验可以分为以下4组：（1）生物素修饰纳米银催化氢醌/硝酸银显色（结果如图6曲线a 所示）；（2）生物素修饰纳米银催化银增强显色（结果如图6曲线b 所示），（3）链亲和素纳米金催化氢醌/硝酸银显色（结果如图7曲线a 所示）；（4）链亲和素纳米金催化银增强显色（结果如图7曲线b ）．综合图6和图7的结果可以发现，实验（1）和（2）的检出限低于实验（3）和（4），说明本实验中修饰状态下的纳米银催化特性强于纳米金．此外，实验（1）的最低检测量及图像背景均低于实验（2），且显色时间也比实验（2）短，说明氢醌/硝酸银作为显色剂的效果强于商品化的银增强试剂．综上所述，本实验基于探针修饰方法的创新及显色剂的优化，设计合成了生物素修饰纳米银探针及氢醌/硝酸银显色试剂，并成功地实现了对蛋白芯片上微量蛋白的灵敏、可视化检测．与商品化的链亲和素纳米金检测探针及银增强显色试剂相比，本文报道的检测试剂具有更高的检测灵敏度（检测灵敏度提高了约40倍），显色速度更快，且探针十分稳定，常温保存即可．参考文献［1］Deng X．Y．，Gao D．J．，Tian Y．，Chen Y．H．，Yu A．M．，Zhang H．Q．，Wang X．H．，Chen Y．．Chem．Res．Chinese Universities［J ］，2010，26（1）：23—26［2］Liu B．H．，Tsao Z．J．，Wang J．J．，Yu F．Y．．Anal．Chem．［J ］，2008，80：7029—7035［3］Liang R．Q．，Tan C．Y．，Ruan K．C．．J．Immunol．Methods ［J ］，2004，285：157—163［4］Duan L．L．，Wang Y．F．，Li S．S．C．，Wan Z．X．，Zhai J．X．．BMC Infectious Diseases ［J ］，2005，5：53［5］Guo H．S．，Zhang J．N．，Yang D．，Xiao P．F．，He N．Y．．Colloid．Surf．B ［J ］，2005，40：195—198［6］Gupta S．，Huda S．，Kilpatrick P．K．，Velev O．D．．Anal．Chem．［J ］，2007，79：3810—3820［7］Taton T．A．，MirkinC．A．，Letsinger R．T．．Science ［J ］，2000，289：1757—1760［8］Wei H．，Chen C．G．，Han B．Y．，Wang E．K．．Anal．Chem．［J ］，2008，80：7051—7055［9］Thompson D．G．，Enright 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bio-AgNPs were employed to couple with the protein via stripavaidin labeled anti-human IgG．The results show that the linear relationship of protein concen-tration is between 160fg and 100pg and the limit of detection is 160fg （S /N =3）．Compared with the method using SA-labeled gold nanoparticle or silver enhancement ，the sensitivity of this method is increased about 40fold．The presented method shows its advantages including high sensitivity ，stability and rapidity．KeywordsBiotinylated silver nanoparticle （Bio-AgNP ）；Hydroquinone /AgNO 3；Micro-protein detection ；Protein chip（Ed．：A ，G ）9812No．11李慧等：生物素修饰纳米银探针的制备及在蛋白芯片可视化检测中的应用。

分子印迹技术在食品安全检测中的应用杨卫海;张吉;夏明星;徐月静;严守雷;王清章【摘要】The basic principle and classification of molecular imprinting technology was introduced; and its application in food safety detection and analysis such as solid-phase exaction, sensor technology and chromatographic analvsis was reviewed.The prospect of this technology was also put forward.%综述了分子印迹技术的基本原理及分类,并对其在固相萃取、传感器、色谱分析等食品安全检测中的应用进行了概述,同时展望了分子印迹技术的应用前景和发展趋势.【期刊名称】《湖北农业科学》【年(卷),期】2011(050)013【总页数】3页(P2598-2600)【关键词】分子印迹技术;食品安全;检测分析【作者】杨卫海;张吉;夏明星;徐月静;严守雷;王清章【作者单位】山东省黄岛出入境检验检疫局,山东,青岛,266555;华中农业大学食品科学技术学院,武汉,430070;山东省黄岛出入境检验检疫局,山东,青岛,266555;山东省黄岛出入境检验检疫局,山东,青岛,266555;山东省黄岛出入境检验检疫局,山东,青岛,266555;华中农业大学食品科学技术学院,武汉,430070;华中农业大学食品科学技术学院,武汉,430070【正文语种】中文【中图分类】TS201.2“民以食为天，食以安为先”。

食品安全是一个重要的民生问题。

食品中有害化学物质残留、非食品添加剂以及病原微生物污染问题仍是当前食品安全最为重要的因素之一。

近年来，我国食品安全问题日益突出，如三聚氰胺毒奶粉事件、瘦肉精事件、海南毒豇豆事件等，这些无不为食品安全敲响了警钟。

中文摘要大葱油注射液对胃癌裸鼠移植瘤的抑瘤作用摘要目的：本实验为中国大葱防治胃癌系列研究的后续研究，前期实验中用大葱提取物作用于体外培养的人胃腺癌细胞株ＭＧＣ．８０３，能有效抑制胃癌细胞增殖，低浓度有诱导细胞分化作用，高浓度则诱导凋亡；证实Ｃａ２＋和ＣＡＭＰ是胃癌细胞凋亡信号传导过程中的重要信息分子，大葱油可能是通过上调细胞内第二信使Ｃａ２＋和ＣＡＭＰ水平实现的。

本实验将大葱油制各成注射液，完成其对人胃腺癌细胞株ＭＧＣ．８０３体外抗肿瘤敏感性检测后；观察该注射液对荷瘤裸鼠ＭＧＣ．８０３皮下移植瘤的生长抑制作用及其毒性作用。

方法：确定大葱油注射液半数杀伤浓度（ＩＣ，。

）：人胃腺癌细胞株ＭＧＣ．８０３复苏后接种于含小牛血清的ＲＭＰｌｌ６４０培养液中，３７℃，５％二氧化碳条件下培养。

取对数生长期细胞，采用ＭＴＴ法，不同浓度的大葱油注射液（１００１ａｇ／ｍｌ、７５１ａｇ／ｍｌ、５０ｒｔｇ／ｍｌ、２５１ａｇ／ｍｌ、１２．５１ａｇ／ｍ１），作用于ＭＧＣ．８０３细胞２４小时、４８小时、７２小时后，测定细胞增殖抑制率，用ＰＯＭＳ软件程序运算出半数杀伤浓度（ＩＣｓｏ）。

大葱油注射液的制备：大葱油０．４９加入２０ｍｇ／ｍｌ的吐温一８０溶液后，安瓿封存。

１００℃流通蒸气灭菌３０分钟，贮存在避光处。

大葱油注射液毒性试验：取６～７周龄，体重２０～２２中文摘要克的昆明种小鼠，每１０只为一组，共两组。

第一组腹腔注射１０ｍｇ／ｍｌ的大葱油注射液０．５ｍｌ，第二组腹腔注射０．２５ｍｌ。

用药后观察一周，未见小鼠死亡，遂行最大耐受量实验。

取６～７周龄，体重２０～２２克的昆明种小鼠２０只，给每只小鼠腹腔注射最大浓度（１０ｍｇ／ｍ１）和最大容积（１ｍ１）的大葱油注射液，上、下午各一次，观察７天。

建立裸鼠移植瘤模型：选用对数生长期的ＭＧＣ－８０３细胞，收集并调整细胞数，用无血清的培养液稀释细胞使细胞终浓度达到１×１０７个／ｍｌ。

引领食品科学研究的前沿期刊食品科学研究是关于食物的生产、加工、质量控制、储存和消费的学科领域。

为了推动食品科学领域的发展，不少期刊致力于引领和报道前沿的研究成果。

本文将介绍几本在食品科学研究领域中具有影响力和领先地位的期刊，并探讨其对研究和学术界的重要意义。

一、《食品化学》（Food Chemistry）《食品化学》是一本国际性的期刊，涵盖了食品中化学成分的研究与分析。

该期刊致力于发表高质量的原创性研究，为食品科学家和化学家提供了一个交流和分享研究成果的平台。

《食品化学》以其内容的广泛性和严谨性而受到学术界的认可，对于引领食品化学研究领域的发展起到了重要作用。

二、《食品微生物学》（Food Microbiology）《食品微生物学》期刊是研究和报道食品中微生物的生态学、毒理学、生理学和分子生物学等方面的前沿成果的重要刊物。

食品微生物是食品安全和卫生的重要因素，因此对其研究和控制具有重要意义。

该期刊的发表文章涵盖了食品微生物学的多个方向，如菌落计数、微生物的特定检测和鉴定方法等。

《食品微生物学》期刊以其对食品安全研究的贡献和对全球食品微生物学发展的引领作用而备受瞩目。

三、《食品工程》（Food Engineering）《食品工程》期刊是关于食品生产和加工工艺的研究领域的重要期刊。

它关注的是食品加工技术和设备，以及食品的质量控制、保鲜、储藏和包装等方面的新技术和新发展。

该期刊的文章主题涵盖了食品工程的多个领域，包括食品加工过程模拟和优化、食品制造业的绿色和可持续发展等。

《食品工程》期刊在推动食品工程领域的发展、提高食品生产和加工效率以及保证食品质量上发挥了重要作用。

四、《食品科学》（Food Science）《食品科学》期刊是涵盖食品科学的各个子学科领域的综合性期刊。

该期刊发表了包括食品的营养学、食品生化学、食品物理学、食品安全等方面的原创研究。

该期刊的文章内容广泛，涉及食物的生产、加工和消费等方面的基础研究和应用研究。