Construction of human liver cancer vascular endothelium cDNA expression library and screening of the endothelium-associated antigen genes  

作　　者：XingZhong Yu-LiangRan Jin-NingLou DongHu LongYu Yu-ShanZhang ZhuanZhou Zhi-HuaYang 

机构地区：[1]DepartmentofCellandMolecularBiology,CancerInstitute(Hospital),ChineseAcademyofMedicalSciencesandPekingUnionMedicalCollege,Beijing,100021,China InstituteofClinicalMedicalSciences,China-JapanFriendshipHospital,Beijing100029,China

出　　处：《World Journal of Gastroenterology》2004年第10期1402-1408,共7页世界胃肠病学杂志（英文版）

基　　金：Supported by the National 863 Program,No.2001AA221251 and the National Natural Science Foundation of China,No.30230150

摘　　要：AIM: To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs)cDNA expression library, so as to find some new possible targets for the diagnosis and therapy of liver tumor.METHODS: HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample, and were cultured and proliferated in vitro. A cDNA expression library was constructed with the mRNA extracted from HLCVECs.Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs, and were then tested for their specificity against HLCVECs and angiogenic effects in vitro, such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells, using immunocytochemistry, immunofluorescence,cell cycle analysis and M-I-l- assays, etc. The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX).The positive clones were sequenced and analyzed by bioinformatics.RESULTS: The primary cDNA library consisted of 2×10^6 recombinants. Thirty-six positive clones were obtained from 6× 10^5 independent dones by immunoscreening. Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes. Among them, 3 were new genes previously unreported, 2 of which were hypothetical genes. The other 15 were already known ones. Series analysis of gene expression (SAGE) database showed that ERP70,GRP58, GAPDH, SSB, $100A6, BMP-6, DVS27, HSP70 and NAC alpha in these genes were associated with endothelium and angiogenesis, but their effects on HLCVECs were still unclear. GAPDH, $100A6, BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries.CONCLUSION: By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs,the functional genes associated with tumor endothelium or angiogenesis were identified. The modified SEREX,xenogeneic functional sAIM:To gain tumor endothelium associated antigen genes from human liver cancer vascular endothelial cells (HLCVECs) cDNA expression library,so as to find some new possible targets for the diagnosis and therapy of liver tumor. METHODS:HLCVECs were isolated and purified from a fresh hepatocellular carcinoma tissue sample,and were cultured and proliferated in vitro.A cDNA expression library was constructed with the mRNA extracted from HLCVECs. Anti-sera were prepared from immunized BALB/c mice through subcutaneous injection with high dose of fixed HLCVECs,and were then tested for their specificity against HLCVECs and angiogenic effects in vitro,such as inhibiting proliferation and inducing apoptosis of tumor endothelial cells,using immunocytochemistry,immunofluorescence, cell cycle analysis and MTT assays,etc.The identified xenogeneic sera from immunized mice were employed to screen the library of HLCVECs by modified serological analyses of recombinant cDNA expression libraries (SEREX). The positive clones were sequenced and analyzed by bio- informatics. RESULTS:The primary cDNA library consisted of 2×10~6 recombinants.Thirty-six positive clones were obtained from 6×10~5 independent dones by immunoscreening.Bio-informatics analysis of cDNA sequences indicated that 36 positive clones represented 18 different genes.Among them,3 were new genes previously unreported,2 of which were hypothetical genes.The other 15 were already known ones.Series analysis of gene expression (SAGE) database showed that ERP70, GRP58,GAPDH,SSB,S100A6,BMP-6,DVS27,HSP70 and NACalpha in these genes were associated with endothelium and angiogenesis,but their effects on HLCVECs were still unclear.GAPDH,S100A6,BMP-6 and hsp70 were identified by SEREX in other tumor cDNA expression libraries. CONCLUSION:By screening of HLCVECs cDNA expression library using sera from immunized mice with HLCVECs, the functional genes associated with tumor endothelium or angiogenesis were identified.The modified SEREX, xenogeneic functional serum screening,was demons

关 键 词：内皮相关抗原基因 肝癌 血管内皮组织 诊断 CDNA文库 

分 类 号：R735.7[医药卫生—肿瘤]