【摘要】 目的:探讨应用RNA激活(RNAa)技术上调p21WAF1/CIP1(p21)表达对人肺癌H441细胞增殖和凋亡的影响。方法:设计靶向抑癌基因p21WAF1/CIP1启动子DNA序列互补的双链RNA分子(dsp21),转染H441细胞后应用半定量逆转录聚合酶链反应(RT-PCR)及蛋白质印迹法检测p21WAF1/CIP1基因mRNA及蛋白质的表达变化,噻唑蓝(MTT)法检测H441细胞增殖速度的变化,流式细胞仪检测H441细胞凋亡率的变化。结果:dsp21转染H441细胞72 h后p21WAF1/CIP1表达显著上调。RT-PCR灰度比值结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1mRNA相对表达量分别为(38.1±2.5)%、(33.5±3.9)%和(96.5±2.3)%,差异有统计学意义,F=87.0,P<0.01;蛋白质印迹法结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1蛋白相对表达量分别为(45.7±2.2)%、(43.2±3.1)%和(93.6±2.5)%,差异有统计学意义,F=79.0,P<0.01;经dsp21作用的H441细胞增殖受到明显抑制,凋亡率较对照组明显升高,F=10.2,P<0.01。结论:RNAa技术激活p21WAF1/CIP1基因表达并抑制肺癌H441细胞增殖,诱导细胞凋亡,为肺癌的基因治疗提供了新的思路和方法。更多还原

【Abstract】 OBJECTIVE: To observe the effects of p21WAF1/CIP1 activation by RNAa on lung cancer cell proliferation and apoptosis.METHODS: A dsRNA(dsp21) targeting the p21WAF1/CIP1 gene promoter at position-322 relative to the transcription start site was transfected into H441 cells.Expression of mRNA and protein was evaluated by semiquantitative RT-PCR and western blot.The H441 cells proliferation was measured by MTT method,and the apoptosis rate was detected by flow-cytometry.RESULTS: The introduction of dsp21 was showed to efficiently upregulate the expression of p21WAF1/CIP1 gene according to the results of RT-PCR and wastern blot,the expressions of blank group,negative group and experiment group were(38.1±2.5)%,(33.5±3.9)% and(96.5±2.3)% by RFPCR(F=87.0,P<0.01),and(45.7±2.2)%,(43.2±3.1)% and(93.6±2.5)% in western blot(F=79.0,P<0.01,as compared with controls).The inhibitory effect on cell proliferation was confirmed by MTT test(P<0.05,as compared with controls).Apoptosis rate of H441 cells treated with dsp21 was significantly higher than that of the control cells(F=10.2,P<0.01).CONCLUSION: dsp21 targeting p21WAF1/CIP1 gene promoter can specifically upregulate the expression of p21WAF1/CIP1 gene in H441 cells,and therefore has an substantially inhibitory effect on cell proliferation in vitro,it provides a new method and material to the gene therapy of lung cancer.更多还原