应用RNAi技术抑制人宫颈癌细胞端粒酶活性表达的研究  

Effects of siRNA on inhibition of hTERT expression of SiHa cells in vitro

作　　者：祁丽[1] 张文轩[1] 邢丽娜[1] 

机构地区：[1]哈尔滨医科大学附属第二医院肿瘤放疗科,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2015年第4期283-287,共5页Journal of Harbin Medical University

基　　金：黑龙江省自然科学基金资助项目(D200974)

摘　　要：目的研究转染前后对SiHa细胞增殖、凋亡及放射敏感性的影响。方法SiHa细胞分为3组：实验组、阴性对照组及空白对照组,其中实验组和阴性对照组分别用siRNA#1～4和阴性对照siRNA转染,空白对照组不做任何转染处理。用实时荧光定量PCR和蛋白印迹法检测各组细胞hTERTmRNA和蛋白的表达,CCK-8法检测细胞的增殖情况,流式细胞仪检测各组细胞凋亡率,克隆形成实验通过单靶多击模型拟合计算D0、Dq值,检测细胞的放射敏感性。结果转染后48h及72h,实验组siRNA#3hTERTmRNA及蛋白表达量较阴性对照组下降,P〈0.05;转染96h后实验组细胞A值0.80±0.09,阴性对照组为1.25±0.11,P=0.0054;转染后实验组早期凋亡率（10.50±0.20）%较阴性对照组（5.80±0.10）%明显增加,P〈0.0001;实验组及阴性对照组D0及Dq值分别为1.53Gy、0.77Gy和2.19Gy、1.31Gy,差异有统计学意义（P〈0.05）。结论siRNA能特异性下调SiHa细胞中hTERTmRNA和蛋白水平的表达,提高细胞的早期凋亡率并抑制细胞增殖活性,增强SiHa细胞的放射敏感性。Objective To study effect of siRNA on the cell growth, apoptosis and radiosensitivity of SiHa cells. Methods SiHa cells were divided to 3 groups. Cells in experimental group were transfected with siRNA#1 -4. Cells in control group were transfected with control siRNA. Blank control group had no transfection. Real-time PCR and Western blot were used to deter- mine the expression of hTERT in SiHa cells. CCK-8 kit was used to detect the cell growth ability. Flow cytometry was used to assess apoptosis rate. The radiosensitivity（ DO, Dq） was calcu- lated with single-hit multi-targets model. Results Forty-eight hours and 72 h after transfec- tion, the hTERT mRNA and protein expression in experimental group were lower than those in control group （P 〈 0. 05 ）. Ninety-six hours after transfection, absorbance values of SiHA cells were 0.80 ±0.09 and 1.25 ± 0. 11 in experimental group and those in control group respectively （P = 0. 0054）. The early apoptosis rate of the two groups were 10. 50%± 0. 20% and 5.80% ± 0. 10% , respectively （ P 〈 0. 0001 ）. The values of DO and Dq of the two groups were 1.53 Gy, 0.77Gy and2. 19 Gy, 1.31 Gy （P〈0.05）, respectively. Conclusion siRNA can specifically down-regulate the expression of hTERT mRNA and protein, enhance the percentage of early apoptosis, decrease ability of cell growth, and enhance radiosensitivity.

关 键 词：SIHA细胞 HTERT RNA干扰 细胞凋亡 放射敏感性 

分 类 号：R737.33[医药卫生—肿瘤]