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目的比较荷载Ki67基因小于扰RNA(Ki67-siRNA)的增殖腺病毒ZD55-Ki67及非增殖腺病毒Ad-Ki67对肾癌细胞的杀伤作用。方法MOI=10的ZD55-Ki67、Ad-Ki67感染肾癌786-0细胞,2d后收集细胞,蛋白印迹法检测E1A表达;逆转录-聚合酶链反应(RT-PCR)、蛋白印迹法、免疫细胞化学法检测Ki67表达;原位末端标记法(TUNEL)检测凋亡。4 d后噻唑蓝(MTT)法检测细胞存活率,7 d后结晶紫染色法检测细胞毒性作用。结果感染ZD55-Ki67的786-0细胞表达E1A,感染Ad-Ki67的细胞不表达E1A。ZD55-Ki67、Ad-Ki67感染的786-0细胞Ki67 mRNA表达率分别为(37.9±2.3)%、(64.1±1.9)%;Ki67蛋白表达率分别为(42.5±2.4)%、(60.1±2.2)%;Ki67染色阳性率分别为(20.8±2.8)%、(32.3±2.5)%;786-0细胞存活率分别为(22.2±3.0)%、(60.4±3.4)%;凋亡率分别为(53.0±3.7)%、(35.3±2.5)%,两种病毒处理之间差异均有统计学意义(P<0.01)。结论ZD55-Ki67抑制786-0细胞Ki67表达及增殖、诱导凋亡及细胞毒性作用均显著优于Ad-Ki67。增殖腺病毒介导的RNA干扰杀伤肾癌细胞作用优于非增殖腺病毒。
OBJECTIVE: To compare the cytotoxicity of Ki67-deficient siRNA against Ki67-siRNA adenovirus ZD55-Ki67 and non-proliferating adenovirus Ad-Ki67 against renal cancer cells. METHODS: Human renal cancer 786-0 cells were infected with ZD55-Ki67 and Ad-Ki67 at MOI = 10. Cells were harvested after 2 days. The expression of E1A was detected by Western blotting. The expression of E1A was detected by RT-PCR, Western blotting, Ki67 expression was detected by cytochemistry; apoptosis was detected by TUNEL. After 4 days, cell viability was detected by MTT assay. Cytotoxicity was detected by crystal violet staining after 7 days. As a result, 786-0 cells infected with ZD55-Ki67 expressed E1A, while cells infected with Ad-Ki67 did not express E1A. The expression rates of Ki67 mRNA in 786-0 cells infected by ZD55-Ki67 and Ad-Ki67 were (37.9 ± 2.3)% and (64.1 ± 1.9)%, respectively. The expression rates of Ki67 protein were (42.5 ± 2.4)% and (60.1 ± 2.2) ), And the positive rate of Ki67 staining was (20.8 ± 2.8)% and (32.3 ± 2.5)% respectively; the survival rates of 786-0 cells were (22.2 ± 3.0)% and (60.4 ± 3.4)% (53.0 ± 3.7)% and (35.3 ± 2.5)%, respectively. The difference between the two viruses was statistically significant (P <0.01). Conclusion ZD55-Ki67 can inhibit Ki67 expression, proliferation, apoptosis and cytotoxicity in 786-0 cells significantly better than Ad-Ki67. Proliferation of adenovirus-mediated RNA interference killing of renal cancer cells than non-proliferating adenovirus.