RNA干扰靶向人端粒酶反转录酶对小儿肝母细胞瘤细胞的生长抑制作用  

Inhibitory effect of RNA interference targeting human telomerase reverse transcriptase against hep- atoblastoma cells

作　　者：夏自强[1] 丁道奎[1] 张宁[1] 杨合英[1] 张大[1] 王家祥[1] 

机构地区：[1]郑州大学第一附属医院儿外科,450052

出　　处：《中华实验外科杂志》2017年第1期113-116,共4页Chinese Journal of Experimental Surgery

摘　　要：目的利用RNA干扰（RNAi）技术降低人端粒酶反转录酶（hTERT）基因的表达，抑制其活性，观察其对小儿肝母细胞瘤细胞的增殖和凋亡影响。方法构建针对hTERT基因的小干扰RNA（siRNA）真核表达载体——pLXSN-增强型绿色荧光蛋白（EGFP）-U6-siTERT，采用聚合酶链反应（PCR）法扩增hTERT-siRNA、EGFP、U6＋27的DNA片段，反转录病毒表达载体pLXSN-EGFP-U6-siTERT的构建及酶切鉴定，制备靶向hTERT的重组反转录病毒；用重组反转录病毒感染HepG2小儿肝母细胞瘤细胞，以抗酒石酸酸性磷酸酶（TRAP）法检测端粒酶活性变化；流式细胞仪检测细胞凋亡率；噻唑蓝（MTT）法检测肿瘤细胞生长抑制。结果成功制备出hTERT-siRNA反转录病毒表达载体；对照组端粒酶活性为2 143.06±198.69，重组病毒感染作用24、48、72 h后，端粒酶活性分别为1 632.02±116.28、899.38±126.11和321.25±25.25，与感染前比较，分别下降23.84%、58.03%和85.01%，各时间组间差异有统计学意义（P＝0.046、0.024和0.008）。流式细胞仪检测结果显示：不同滴度的重组病毒感染实验组与对照组的细胞凋亡率比较差异有统计学意义，并呈浓度依赖关系，随着滴度的增加细胞凋亡率增加。1×105菌落形成单位（CFU）重组病毒感染24 h后，其凋亡率达29.05%。MTT结果显示，感染后24 h细胞凋亡率为29.05%；重组病毒感染后24 h，肿瘤细胞开始明显死亡，随着病毒滴度的提高和作用时间延长，细胞死亡亦逐渐加重，病毒滴度为6.0×105 CFU时，感染24、48、72 h后，MTT结果为0.29±0.14、0.20±0.13和0.18±0.11，3.0×105 CFU时，感染24、48、72 h后，MTT结果为0.32±0.11、0.26±0.12和0.25±0.10，1.0×105 CFU时，感染24、48、72 h后，MTT结果为0.33±0.12、0.26±0.13和0.26±0.12，1.0×104 CFU时，感染24、48、72 h后，MTT结果为0.43±0.14、0.35±0.10和0.33±0.15，1.0×103 CFU时，感染24、48、72 h后，MObjective To construct a recombinant retrovirus vector expressing small interfering RNA （siRNA） targeting human telomerase reverse transcriptase （hTERT）, and assess its effect on proliferation and apoptosis of human hepatoblastoma cells.Methods The sequences of the siRNA targeting hTERT, U6 promoter and enhanced green fluorescent protein （EGFP） gene were amplified by polymerase chain reaction （PCR） and inserted into the mammalian retroviral expression vector pLXSN. The PCR method was used to amplify hTERT-siRNA, EGFP, DNA fragment of U6＋ 27. The retroviral expression vector pLXSN-EGFP-U6-siTERT was constructed and subjected to enzyme digestion identification. The recombinant retroviral vector pLXSN-EGFP-U6-siTERT was constructed. The vector was then used to infect human hepatoblastoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by methyl thiazol tetrazolium （MTT） assay.Results hTERT-siRNA retroviral expression vectors were successfully prepared. In the control group, the telomerase activity was 2 143.06±198.69. At 24, 48 and 72 h after colony forming units （CFU） recombinant virus infection the telomerase activity was 1 632.02±116.28, 899.38±126.11 and 321.25±25.25 respectively. As compared with before infection, the telomerase activity was respectively decreased by 23.84%, 58.03% and 85.01%. There was significant difference among different time groups （P=0.046, 0.024 and 0.008）. The results of flow cytometry showed that there was significant difference in the cell apoptosis rate between the experimental group and the control group with different titer of recombinant virus infection, and there was a concentration-dependent relationship. At 24 h after 1×105 CFU recombinant virus infection, the apoptosis rate was 29.05%. MTT results showed that apoptosis rate was 29.05% at 24 h

关 键 词：RNA干扰 人端粒酶反转录酶 肝母细胞瘤 脱噬作用 

分 类 号：R735.7[医药卫生—肿瘤]