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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: To make gene expression of exogenous TNF, IFN genes and endogenous major histocompatibility complex class I (MHC I) positive feedback. METHODS: The adenovirus vector was used to ligate TNF and IFN genes with IRES. HLA B7-inducible promoter was chosen to regulate its expression. The transcription and protein expression of transduction genes were detected by RT PCR, antibody neutralization assay and MTT assay respectively. The expression of MHC class I molecules was detected by flow cytometry. Results: The TNFα and IFNβ genes were efficiently transcribed in human hepatocellular carcinoma cells. The expression of MHC class I molecules in transfected cells (SMAdBT1, SMAdCTⅠ) was higher than that in SMAd control group, and SMAdBT1 group was significantly higher than SMAdC TI group. The highest activity of TWF: SMAdBTI and SMAd were 1059ng ml and 415pg ml, respectively. The highest IFN activity was 2 987U 2 4h 10 6 cells and 2 6262U 2 4h 10 6 cells respectively. Anti-TNFα polyclonal antibody and anti-IFNβ polyclonal antibody both had neutralizing activity on SMAdBTI and SMAdCTI supernatant. CONCLUSION: Human TNFα and IFNβ genes regulated by adenovirus-mediated HLA B7 inducible promoter can be expressed in human hepatocellular carcinoma cell line SMMC 772 1, which can significantly increase the expression of TNF, IFN and MHC class I molecules.