慢病毒介导的CRYAB基因沉默对胃癌细胞SGC-7901增殖和迁移能力的影响

发布时间：2018-05-30 20:39

本文选题：CRYAB基因 + 慢病毒　；参考：《兰州大学》2017年硕士论文

【摘要】：目的本研究采用慢病毒介导的RNA干扰技术,通过体外实验研究CRYAB(alphaβ-crystallin,晶状体蛋白αβ)基因沉默对胃癌细胞SGC-7901增殖与迁移能力的影响。方法1.构建靶向CRYAB基因特异性shRNA慢病毒载体并转染人胃癌细胞SGC-7901细胞株,荧光显微镜观察拍照检测转染效率;2.Real-time PCR和Western Blotting方法检测沉默CRYAB基因后CRYAB的mRNA和蛋白质水平;3.CCK-8法与细胞克隆球形成实验检测沉默CRYAB基因后细胞的增殖活性;4.Transwell迁移实验与细胞划痕实验检测沉默CRYAB基因后细胞的迁移能力。结果1.荧光显微镜观察结果显示,感染复数(MOI)值为20的SGC-7901细胞的病毒转染率大于80%;2.Real-time PCR与Western Blotting结果显示,成功转染sh RNA-CRYAB胃癌细胞SGC-7901细胞株。慢病毒转染72h后,与sh-ctrl组比较,sh-CRYAB组CRYAB mRNA水平减少90%;与sh-ctrl组比较,sh-CRYAB组CRYAB的蛋白表达显著降低(P0.001);3.细胞克隆球形成实验显示,SGC7901细胞感染shRNA慢病毒的细胞克隆数明显较感染对照慢病毒组的克隆数少,两组比较具有统计学意义(P0.05);4.CCK-8检测结果显示,SGC-7901细胞的增殖能力于CRYAB基因沉默之后显著减弱。与sh-ctrl组相比,sh-CRYAB组细胞增长速度变缓,在第4天、第5天增殖能力降低(P0.05,P0.01)。5.Transwell小室细胞迁移实验结果显示,转染CRYAB shRNA SGC-7901细胞的迁移能力明显下降。sh-CRYAB组平均穿膜细胞数是(49.5±2.0),sh-ctrl组平均穿膜数为(370.0±2.9),两组数据具有统计学差异性(P0.01);6.细胞划痕实验结果显示,sh-ctrl组划痕基本被迁移的细胞覆盖,sh-CRYAB组的划痕仍比较清楚,测量两组0、24、48、72 h的相对空白面积,sh-CRYAB组细胞相对空白面积在24、48、72 h小于sh-ctrl组(P0.01)。结论shRNA-CRYAB慢病毒干扰载体对抑制CRYAB基因于胃癌细胞SGC-7901中发生机制具有显著作用,进一步使细胞增殖与迁移受到抑制。CRYAB有望成为肿瘤基因治疗的新靶向分子。
[Abstract]:Objective to study the effect of CRYAB (alpha beta -crystallin, crystallin alpha beta) gene silencing on the proliferation and migration of gastric cancer cell SGC-7901 in vitro by using lentivirus mediated RNA interference. Method 1. construction of targeted CRYAB gene specific shRNA lentivirus carrier and transfection of human gastric cancer cell SGC-7901 cell lines. The transfection efficiency was detected by microscope, and the mRNA and protein levels of CRYAB after the silent CRYAB gene were detected by 2.Real-time PCR and Western Blotting. The proliferation activity of the cells after the silencing of the CRYAB gene was detected by the 3.CCK-8 method and the cell cloned ball formation test, and the 4.Transwell Migration Experiment and the cell scratch test were used to detect the silenced CRYAB gene. Results 1. fluorescence microscopy showed that the virus transfection rate of SGC-7901 cells with complex number (MOI) value of 20 was more than 80%. The results of 2.Real-time PCR and Western Blotting showed that the SGC-7901 cell line of SH RNA-CRYAB gastric cancer cells was successfully transfected. Compared with group sh-ctrl, the protein expression of CRYAB in group sh-CRYAB was significantly lower than that in group sh-ctrl (P0.001), and the 3. cell cloned ball formation experiment showed that the number of clones of SGC7901 cells infected with shRNA lentivirus was less than that of the infection control lentivirus group, and the two groups were statistically significant (P0.05); 4.CCK-8 detection results showed SGC-7901 fine. The cell proliferation ability decreased significantly after the CRYAB gene silencing. Compared with the sh-ctrl group, the cell growth rate of sh-CRYAB group slowed, and the proliferation ability decreased on the fourth day, fifth days (P0.05, P0.01).5.Transwell cell migration experiment showed that the transfer ability of CRYAB shRNA SGC-7901 fine cell decreased significantly in the.Sh-CRYAB group. The number of cells was (49.5 + 2), the average membrane number of the sh-ctrl group was (370 + 2.9), and the two groups had statistical difference (P0.01). The results of the 6. cell scratch test showed that the scratches in the sh-ctrl group were basically covered by the migrated cells, the scratches in the sh-CRYAB group were still clearer, the relative blank area of the two groups of 0,24,48,72 h was measured, and the cells of the sh-CRYAB group were relatively blank. The area of 24,48,72 h is less than group sh-ctrl (P0.01). Conclusion shRNA-CRYAB lentivirus interference carrier plays a significant role in the mechanism of inhibiting the occurrence of CRYAB gene in gastric cancer cell SGC-7901. The further inhibition of cell proliferation and migration is expected to be a new target for tumor gene therapy.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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