SurvivinshRNA-APC双基因共表达慢病毒载体对HT-29结肠癌细胞裸鼠皮下移植瘤生长情况的影响

发布时间：2018-05-17 12:15

本文选题：皮下移植瘤 + 双基因共表达慢病毒载体　；参考：《医学研究生学报》2017年06期

【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(P0.05);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(P0.05),而体积抑制率、瘤重抑制率均增加(P0.05);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(P0.05)。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(P0.05);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(P0.05)。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(P0.05),而APC组、Survivin shRNA组、双基因组亦较空载组[(10.06±0.43)%]明显升高(P0.05);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(P0.05)。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。
[Abstract]:Objective RNA interference at post-transcriptional level has been widely used in multi-gene therapy. The aim of this study was to establish a subcutaneous xenograft model of human HT-29 colon cancer cells in nude mice, and to study the effect of Survivin shRNA-APC double gene co-expression lentivirus vector on the growth of human colon cancer xenografts in nude mice by combining RNAi and exogenous supplementation. Methods Thirty-five nude mice were randomly divided into five groups, namely, Survivin shRNA-APC double-genome survivin shRNA group, no-load group and blank group with 7 rats in each group, and Survivin shRNA-APC-survivin shRNA and APC stable transformants were coexpressed in logarithmic growth phase. Human HT-29 colon cancer cells were subcutaneously transplanted into the left anterior axillary region of 5 groups of nude mice, and the tumor volume and weight were measured by measuring the volume and weight of human colon cancer cell line (HT-29), which was injected into 5 groups of nude mice respectively by injection of the no-load stable transformation strain (2 脳 10 6 / mL) into the subcutaneous xenograft model of human colon cancer cells. Real time PCR was used to detect the expression of survivin mRNA and the expression of Survivin protein was detected by immunohistochemistry. Tunel was used to detect apoptosis. Results compared with the control group, the average volume and average weight of the tumor were reduced by P0.05, and the mean volume and the mean weight of the tumor were also decreased by P0.05 and volume inhibition rate in the shRNA group compared with the control group, and the mean volume and the mean weight of the tumor were also decreased in the shRNA group compared with the control group, and the volume inhibition rate of the tumor was also decreased compared with the control group. The tumor weight inhibition rate was increased by P0.05G, and the mean volume and weight of the transplanted tumor were increased in the APC group and the survivin shRNA group as compared with those in the double genome group (P 0. 05%, P 0. 05%, P 0. 05%). Compared with the control group and the no-load group, the relative content of Survivin mRNA and protein in the survivin shRNA group was significantly lower than that in the Survivin shRNA group, and that in the Survivin shRNA group was significantly lower than that in the Survivin shRNA group. Compared with the control group, the apoptotic index of colon cancer cells in transplanted tumor tissue of nude mice [9.89 卤0.31%] was significantly higher in APC group than in survivin shRNA group [31.19 卤1.79%, 33.64 卤2.03%, 56.72 卤3.17%], while in APC group, survivin shRNA group was significantly higher than that in APC group, while in the control group, the apoptosis index of colon cancer cells was 9.89 卤0.31%, while that in APC group was significantly higher than that in survivin shRNA group. Compared with the control group, the apoptotic index of survivin shRNA-APC was significantly higher than that of the control group [10. 06 卤0. 43%], and the apoptosis index of survivin shRNA-APC tumor cells was significantly higher than that of the Survivin shRNA group (P 0. 05%, P 0. 05%, P 0. 05%, P 0. 05%, P 0. 05%, P 0. 05%). Conclusion Survivin shRNA-APC double gene co-expression lentivirus vector can reduce the expression level of Survivin gene, promote the apoptosis of colon cancer cells, inhibit the growth of transplanted tumor, and is superior to the effect of single gene.
【作者单位】：佳木斯大学附属第一医院消化二科;佳木斯大学临床医学院;
【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)
【分类号】：R735.35

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