腺病毒介导HLAB-7可诱导启动子调控双细胞因子基因在人肝癌细胞中表达

Adenoviral vector-mediated transfer of huTNFα and huIFN β gene in human hepatocellular carcinoma cells controlled by HLA-B7 inducible promoter

目的 :使外源性TNF、IFN基因和内源性主要组织相容性复合物Ⅰ类 (MHCⅠ )分子基因表达呈“正反馈”式放大效应。方法 :以腺病毒为载体 ,以IRES连接TNF和IFN基因 ,选择HLA B7可诱导启动子调控其表达。分别用RT PCR、抗体中和实验和MTT法检测转导基因的转录及蛋白表达 ,流式细胞仪检测MHCⅠ类分子表达。结果 :TNFα和IFNβ基因在人肝癌细胞中可有效转录 ,转染细胞 (SMAdBT1、SMAdCTⅠ )的MHCⅠ类分子表达高于SMAd对照组 ,且SMAdBT1 组明显高于SMAdC TI组。TWF最高活性 :SMAdBTI和SMAd分别为 1 0 5 9ng ml、4 15pg ml;IFN最高活性分别为 2 98 5 7U 2 4h 10 6 个细胞和 2 6 2 2 6U 2 4h 10 6 个细胞。抗TNFα多抗、抗IFNβ多抗对SMAdBTI、SMAdCTI上清均具有中和活性。 结论 :腺病毒介导的HLA B7可诱导启动子调控的人TNFα和IFNβ基因导入人肝癌细胞株SMMC 772 1,可明显增加其对TNF、IFN和MHCⅠ类分子的表达。

Objective:INF α and IFN β gene fragment was introduced into human hepatocellular carcinoma cell line SMMC-7721 to increase tumor immunogenecity by enhancing gene expression of TNF,IFN and MHC class I molecule.Methods:Recombinant adenovirus was used as vector,TNFα and IFNβ were connected through IRES and HLA-B7 promoter was selected to control their expression.RT-PCR and MTT were used to detect the genes′ transcription and protein expression,MHC class I molecule expression was measured by FACS assays.Results:The genes of TNFα and IFNβ were effectively transcripted.MHC class I molecule in study groups was significantly higher than that of control group.TNFα activity was 298.57 U/24 h/10 6 cells and 262.26 U/24 h/10 6 cells respectively.Antibody neutralization experiment showed that transfected cells could generate the proteins of TNFα and IFNβ.Conclusion:The transfected human hepatocelullar carcinoma cells had an elevated production of TNF,IFN and MHC Ⅰ.