RNAi沉默人端粒酶逆转录酶对HeLa细胞生长及药物敏感性影响的研究  

Effect of silencing hTERT by RNAi on growth and drug sensitivity of HeLa cells

作　　者：米洋[1] 廖旻晶[1] 郭雪玲[1] 黄喜阳[1] 黄小荣[1] 梁昌盛[1] 周俊宜[1] 

机构地区：[1]中山大学中山医学院生化教研室,广东广州510080

出　　处：《中华肿瘤防治杂志》2009年第11期829-833,共5页Chinese Journal of Cancer Prevention and Treatment

基　　金：广东省自然科学基金重点项目(04105351);广东省科技计划项目(2006B35502007)

摘　　要：目的:探讨利用短发夹RNA(shorthairpin RNA,shRNA)抑制人端粒酶逆转录酶(hTERT)的表达后对人宫颈癌HeLa细胞生长及药物敏感性的影响。方法:将构建的hTERT-shRNA真核表达载体用脂质体转染入HeLa细胞中,经G418筛选得到稳定转染的细胞株,并绘制细胞生长曲线。RT-PCR和蛋白质印迹法分别检测hTERT、c-myc mR-NA和蛋白表达。镜下观察干扰组细胞与对照组细胞对化疗药物的反应,并在紫杉醇给药后采用流式细胞术和细胞免疫化学的方法分别检测两组细胞的死亡率和细胞内微管组织状态。结果:干扰组细胞在6代以后hTERT的mRNA和蛋白表达水平均显著低于对照组,P<0.05,n=3,而c-myc的表达以及细胞的生长速度与对照组差异无统计学意义,P>0.05,n=3。干扰组细胞对化疗药物紫杉醇的反应明显不同于对照组,紫杉醇给药后干扰组细胞的死亡率(9.7%)高于对照组细胞(6.4%),但给药前后两组细胞内微管的分布差异无统计学意义,P>0.05,n=3。结论:单独下调HeLa细胞中hTERT的表达不能降低肿瘤细胞的生长速度,也不能显著改变c-myc的表达,却可以增加细胞对紫杉醇的敏感性,而hTERT是否参与微管的调节有待进一步探讨。OBJECTIVE： To explore the effect of silencing short hairpin RNA （shRNA） targeting hTERT on HeLa cells. METHODS： Expression vectors generating shRNA targeting hTERT were constructed and introduced into HeLa cells by lipo some. Stable cell strains were gained by G418 screening and their growth velocities were measured. The expression of hTERT and e-myc were analyzed by RT-PCR and Western blot. The response of hTERT silencing cells and the control group to chemotherapeutic drugs was observed. Flow cytometry and im- munochemistry were used to analyze the cell death rate and the organization of mierotubule after the two groups were treated with paclitaxel. RESULTS： The mRNA and protein expressions of hTERT in the sixth generation of hTERT-silencing cells were less than those of the control group （P〈0.05, n 3）, but the expression of c-mye and cell growth velocity had no significant difference （P） 0.05, n = 3）. The response to paclitaxel of hTERT-silencing cells was quite different from that of the con- trol group. After paclitaxel treatment, the death rate of hTERT silencing cells （9.7 % ） was higher than that of the control group （6.4%）, but no significant difference was found between these two groups in the organization of microtubule （P〉0.05, n=3）. CONCLUSIONS： hTERT silencing does not inhibit the growth of HeLa cells or induce changes in c-myc expression, but renders more sensitivity to HeLa cells in the paclitaxel treatment. Whether hTERT takes part in the organization of microtubules to bring a dif- ferent drug response remains to be further explored.

关 键 词：末端转移酶 HTERT RNA干扰 基因 C-MYC 微管 

分 类 号：R737.33[医药卫生—肿瘤]