RNA干扰沉默人端粒酶逆转录酶基因治疗大肠癌移植瘤  

Treatment of transplanted tumor from colotectal cancer in nude mice by RNA interference silencing human telomerase reverse transcriptase gene

作　　者：葛莲英[1] 刘爱群[1] 罗小玲[1] 蔡艳玲[1] 耿芳芳[1] 

机构地区：[1]广西医科大学附属肿瘤医院内镜室,南宁530021

出　　处：《中华实验外科杂志》2012年第10期2012-2014,共3页Chinese Journal of Experimental Surgery

基　　金：广西卫生厅重点科研项目（重00971）;广西自然科学基金资助项目（2010GXNSFA013238）

摘　　要：目的观察重组质粒pGPU6／绿色荧光蛋白（GFP）／Neo-人端粒酶逆转录酶（hTERT）短发卡RNA（shRNA）对SW480细胞裸鼠移植瘤的生长抑制作用。方法将大肠癌SW480细胞（约5×10^7个）接种于裸鼠，成瘤后将裸鼠分3组，每组8只，瘤体内分别注射20μg pGPU6／GFP／Neo—hTERTshRNA（实验组）、20I-LgpGPU6／GFP／Neo—NCshRNA（阴性对照组）、100μl生理盐水（空白对照组）。观察各组裸鼠体内瘤体生长速度，绘制肿瘤生长曲线，采用聚合酶链反应（PCR）、免疫组织化学技术测定肿瘤组织hTERT表达水平，PCR-端粒重复扩增程序（TRAP）-酶联免疫吸附试验（ELISA）法检测端粒酶活性，原位末端标记技术（TUNEL）检测细胞凋亡。结果实验组较其他对照组裸鼠移植瘤生长缓慢，hTERTmRNA的相对表达量为0．388±0．093，hTERT蛋白表达的平均灰度值为171．42±30．94，表达水平均明显低于其他对照组（P〈0．05）；实验组端粒酶活性吸光度（A）值A450-A630为0．484±0．359，较其他对照组明显降低（P〈0．05或P〈0．01）；凋亡指数为（36．30±5．05）％，较其他对照组明显降低（P〈0．01）。结论靶向hTERT基因的shRNA瘤内注射能延缓大肠癌细胞SW480裸鼠皮下移植瘤的生长，其机制可能是通过抑制hTERT mRNA和蛋白的表达，抑制端粒酶的活性，促进肿瘤细胞凋亡而发挥作用。Objective To study the effect of short hairpin RNA （shRNA） fragment of human telomerase reverse transcriptase （pGPU6/GFP/Neo-hTERT shRNA） on the growth of transplanted tumors of colorectal carcinoma in nude mice in vivo. Methods Human colorectal carcinoma cell line 5×10^7 SW480 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were randomly divided into the following 3 groups, 8 nude mice each group. The experimental group was injected with hTERT-shRNA plasmid （20 μg） intratumoraly. NC-shRNA group （20 μg） and NS group （100 μl） served as negative group and blank group, respectively. The tumor growth was observed, and the cuve of tumor growth was drawn. The collected tumor specimens were examined, and the expression of hTERT mRNA and protein was detected. Telomerase activity and cell apoptosis were assayed. Results The trans- planted tumor in experimental group grew more slowly than negative control and blank control groups. The relative expression of hTERT mRNA in experimental group was 0. 388 ± 0. 093 and the average gray value of hTERT protein was 171.42 ±30. 94, which were significantly lower than in control groups （ P 〈 0. 05, P 〈 0. 01, respectively）. The activity of telomerase （ A450-A630 ） in experimental group （ 0. 484 ± 0. 359 ） was markedly lower than in control groups （P 〈 0. 05, P 〈 0. 01, respectively）. The proportion of apoptotic ceils in experimental group （36. 30 ±5.05 ） % was higher than in control groups （ P 〈 0. 01 ）. Conclusion The shRNA fragment of hTERT gene can partly inhibit the growth of SW480 transplantrf tumor in nude mice probably by suppressing hTERT mRNA and protein expression and the activity of telomerase, as well as in- ducing apoptosis of SW480 cells.

关 键 词：RNA干扰 端粒酶逆转录酶 移植瘤 

分 类 号：R735.34[医药卫生—肿瘤]