GPSM2真核表达载体构建及对人胰腺癌细胞迁移能力的影响

发布时间：2018-05-18 05:30

本文选题：胰腺肿瘤 + GPSM2　；参考：《江苏大学》2017年硕士论文

【摘要】：研究目的:本实验通过构建G蛋白信号调节蛋白2(G-p roteinsignaingmodulator2,GPSM2)稳定高表达的胰腺癌细胞株,研究GPSM2与人胰腺癌细胞迁移能力的关系。探讨以GPSM2作分子靶点,为胰腺癌的治疗提供新思路和新策略。研究方法:1、构建GPSM2基因过表达质粒,并转染人胰腺癌MIA-PaCa-2细胞株。2、采用RT-PCR技术检测GPSM2基因转染组、未转染组(空白对照组)和空载体p C M V-Tag3B转染组(阴性对照组)中GPSM2基因m R N A的表达水平。3、Western-blot检测各组胰腺癌细胞GPSM2、β-连环蛋白(β-catenin)的表达。4、采用Transwell实验检测各组胰腺癌细胞迁移能力变化。研究结果:1、成功构建了GPSM2稳定高表达的重组细胞株。2、RT-PCR结果显示GPSM2基因转染组的m R N A表达量较阴性对照组及空白对照组明显增加(均P0.0 1),与空白对照组相比,GPSM2基因上调效率达到了7 3.3倍。阴性对照组与空白对照组间GPSM2mRNA表达水平差异无统计学意义(P0.0 5)3、Western-blot结果证实GPSM2基因重组细胞株中GPSM2蛋白、β-catenin蛋白均高表达(均P0.0 5)。阴性对照组与空白对照组间GPSM2蛋白及β-catenin蛋白表达水平差异无统计学意义(均P0.0 5)。采用一元线性回归分析得出胰腺癌细胞中GPSM2与β-catenin的表达水平呈正比(P0.0 5)。4、Transwel实验发现GPSM2稳定高表达的胰腺癌细胞株的迁移细胞计数明显高于阴性对照组及空白对照组(均P0.0 1)。结论:GPSM2稳定高表达的胰腺癌细胞株的迁移细胞计数明显增加,在人胰腺癌细胞中上调GPSM2的表达能使β-catenin蛋白高表达,可能由此促进人胰腺癌细胞的迁移能力。
[Abstract]:Objective: to study the relationship between GPSM2 and the migration ability of human pancreatic cancer cells by constructing a stable high expression pancreatic cancer cell line 2(G-p rotein signal modulator 2 (GPSM2). To explore the use of GPSM2 as a molecular target to provide new ideas and strategies for the treatment of pancreatic cancer. Methods: GPSM2 gene overexpression plasmid was constructed and transfected into human pancreatic cancer MIA-PaCa-2 cell line. The RT-PCR technique was used to detect the GPSM2 gene transfection group. The expression level of GPSM2 gene m R N A in untransfected group (blank control group) and empty vector p C M V-Tag3B transfection group (negative control group). The expression of GPSM2 gene m R N A in pancreatic cancer cells of each group was detected by Western blot. The expression of GPSM2 and 尾 -catenin was detected by Transwell assay. The migration ability of pancreatic cancer cells in group B was changed. The results showed that the expression of m R N A in the transfected GPSM2 gene group was significantly higher than that in the negative control group and the blank control group (all P0.01, compared with the blank control group). The results of RT-PCR showed that the expression of m R N A in the GPSM2 gene transfected group was significantly higher than that in the negative control group and the blank control group (all P0.01, compared with the blank control group). The efficiency of gene up-regulation was 73.3 times. There was no significant difference in the expression of GPSM2mRNA between the negative control group and the blank control group. The results of Western-blot showed that the expression of GPSM2 protein and 尾 -catenin protein in the recombinant cell line of GPSM2 gene were all high (all P0.05). There was no significant difference in the expression of GPSM2 protein and 尾 -catenin protein between the negative control group and the blank control group (P 0.05). Univariate linear regression analysis showed that the expression of GPSM2 and 尾 -catenin in pancreatic cancer cells was directly proportional to that of P0.05N. 4. Transwell assay showed that the number of migrating cells in pancreatic cancer cell lines with stable and high GPSM2 expression was significantly higher than that in negative control group and blank control group (all P 0.01). Conclusion the migration cell count of human pancreatic cancer cell lines with stable and high expression of GPSM2 increased significantly. Upregulating the expression of GPSM2 in human pancreatic cancer cells may lead to the overexpression of 尾 -catenin protein, which may promote the migration ability of human pancreatic cancer cells.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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