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目的 :研究P5 3反义RNA对肝癌细胞中突变型P5 3致癌性的抑制效应 ,为肝细胞癌基因治疗提供新的方法。方法 :2 .1的人全长cDNA用EcorRⅠ和XbaⅠ的双酶切得到 16 94bp长cDNA片段 ,反向插入哺乳动物表达载体PCR3.1,得到P5 3反义表达载体PCR3.1-P5 3(AS) ,并将其导入人肝细胞株Bel- 74 0 2 (内源性突变型P5 3) ,采用MTT法和FCM方法测定PCR3.1-P5 3(AS)表达的P5 3反义RNA对Bel- 74 0 2细胞生长的影响。结果 :带有PCR3.1-P5 3(AS)的Bel- 74 0 2细胞 ,与对照组Bel- 74 0 2细胞和带PCR3.1空载体的Bel-74 0 2细胞比 ,增殖速度下降 ,FCM的结果也证明实验组细胞大部分受阻于G0 /G1期 ,与细胞对照组及载体对照组有显著的差异。结论 :P5 3反义RNA可有效地抑制肝细胞癌中突变型P5 3的致癌性 ,可用于肝细胞癌实验性基因治疗研究。
Objective: To study the inhibitory effect of P5 3 antisense RNA on the oncogenicity of mutant P5 3 in hepatocellular carcinoma cells, and to provide a new method for gene therapy of hepatocellular carcinoma. Methods: The full-length cDNA of 2.1 was digested with EcoRⅠand XbaⅠ to obtain 1694bp cDNA fragment and inserted into mammalian expression vector PCR3.1 to obtain the P5 3 antisense expression vector PCR3.1-P5 3 ( AS) was introduced into human hepatocyte cell line Bel-74 0 2 (endogenous mutant P5 3). The P5 3 antisense RNA of PCR3.1-P5 3 (AS) was detected by MTT and FCM Bel-74 0 2 cell growth. RESULTS: Bel-7402 cells with PCR3.1-P5 3 (AS) had a slower rate of proliferation than Bel-74 0 2 cells with control and Bel-74 0 2 cells with PCR3.1 empty vector, The results of FCM also showed that most of the cells in the experimental group were blocked in the G0 / G1 phase, which were significantly different from the cell control group and the vector control group. CONCLUSION: P5 3 antisense RNA can effectively inhibit the carcinogenicity of mutant P5 3 in hepatocellular carcinoma and can be used in experimental gene therapy of hepatocellular carcinoma.