摘要
目的在转录后水平进行干预的RNA干扰技术在多基因治疗中的应用越来越广泛。文中旨在构建人HT-29结肠癌细胞裸鼠皮下移植瘤模型,通过联合RNAi和外源性补充的方式研究Survivin shRNA-APC双基因共表达慢病毒载体对结肠癌裸鼠移植瘤生长情况的影响。方法选取35只裸鼠随机数字表法分为5组,即Survivin shRNA-APC双基因组、Survivin shRNA组、APC组、空载组和空白组,每组7只;将已经构建好的处于对数生长期的双基因共表达Survivin shRNA-APC、Survivin shRNA及APC稳转株,空载稳转株,HT-29结肠癌细胞(均为2×106/mL)分别注射至5组裸鼠左前腋下成瘤,构建人HT-29结肠癌细胞裸鼠皮下移植瘤模型;通过测量瘤体体积、重量,检测移植瘤生长抑制率,Real time PCR检测移植瘤组织survivin mRNA的表达,免疫组化检测Survivin蛋白的表达,TUNEL检测凋亡情况。结果与空白组比较,APC组、Survivin shRNA组、双基因组移植瘤平均体积、平均重量均减少(P<0.05);与空载组比较,APC组、Survivin shRNA组、双基因组平均体积、平均移植瘤重量亦减少(P<0.05),而体积抑制率、瘤重抑制率均增加(P<0.05);与双基因组比较,APC组、Survivin shRNA组移植瘤平均体积、平均重量均增加(P<0.05)。与空白组和空载组相比,APC组、Survivin shRNA组、Survivin shRNA-APC双基因组移植瘤组织Survivin mRNA和蛋白表达相对含量明显降低(P<0.05);与Survivin shRNA组、APC组相比,双基因组移植瘤组织Survivin mRNA和蛋白表达相对含量亦明显降低(P<0.05)。与空白组裸鼠移植瘤组织结肠癌细胞凋亡指数[(9.89±0.31)%]比较,APC组、Survivin shRNA组、双基因组[(31.19±1.79)%、(33.64±2.03)%、(56.72±3.17)%]明显升高(P<0.05),而APC组、Survivin shRNA组、双基因组亦较空载组[(10.06±0.43)%]明显升高(P<0.05);与Survivin shRNA组、APC组相比,Survivin shRNA-APC双基因组移植瘤组织癌细胞凋亡指数明显升高(P<0.05)。结论 Survivin shRNA-APC双基因共表达慢病毒载体能够使Survivin基因的表达水平降低,促进结肠癌细胞的凋亡,抑制移植瘤的生长,且优于单个基因的影响。
Objective Post-transcription RNA interference （RNAi） is more and more widely applied in muhigene therapy. This study aimed to establish an subcutaneous xenotransplanted tumor （SXT） model of human HT-29 colon carcinoma in nude mice and in- vestigate the effects of the survivin shRNA-APC double gene co-ex- pression lentiviral vector on the growth of SXT. Methods Thirty- five nude mice were equally divided into five groups, double-gene survivin shRNA, survivin shRNA, APC, empty vector, and blank, and injected into the left anterior axillary with respective stably trans- fected cell lines and human HT-29 colon carcinoma cells, all at 2 ×10^6/mL, to establish an SXT model of human HT-29 colon carcinoma. The inhibition rate of tumor growth was calculated by measuring the size and weight of the SXT, the expressions of survivin mRNA and protein in the tumor tissue detected by real time PCR and immu- nohistoehemistry respectively, and the apoptosis of the HT-29 colon carcinoma cells determined by TUNEL. Results The mean size and weight of the SXT were significantly reduced in the double-gene survivin shRNA-APC, survivin shRNA, and APC groups as com- pared with the blank and empty vector groups （P〈0.05） , though increased in the survivin shRNA and APC groups in comparison with the double-gene group （P〈0.05）. The expressions of survivin mRNA and protein in the tumor tissue were remarkably lower in the double-gene survivin shRNA-APC, survivin shRNA, and APC groups than in the blank and empty vector groups （P〈0.05） , even low- er in the double-gene group than in the survivin shRNA, and APC groups （P〈0.05）. The apoptosis rate of the HT-29 colon carcinoma cells was markedly up-regulated in the double-gene survivin shRNA-APC （[ 56.72±3.17] % ）, survivin shRNA （[ 33.64±2.03] %）, and APE groups （ [ 31.19± 1.791% ） as compared with the blank （ [ 9.89±0.31 ] %） and empty vector groups （ [ 10.06±0.43 ] % ） （P〈 0.05） , even more significantly in the double-gene than in the survivin shRNA and APC groups （P〈0.05）. Conclusion The sur- vivin shRNA-APC double gene co-expression lentiviral vector can reduce the expression level the survivin gene, promote the apoptosis of colon carcinoma cells, and suppress the growth of the subcutaneous xenotransplanted tumor.
出处
《医学研究生学报》
CAS
北大核心
2017年第6期584-590,共7页
Journal of Medical Postgraduates
基金
黑龙江省自然科学基金(H201368)
2015年佳木斯大学研究生科技创新项目(LZZ2015_018)
2016年佳木斯大学研究生科技创新项目(YZ2016_025
YM2016_022)
