【摘要】 目的构建PLCE基因的shRNA表达载体转染膀胱癌细胞T24株,观察其对T24细胞转移侵袭等恶性行为的影响。方法设计合成PLCE基因mRNA的寡核苷酸序列,插入绿色荧光蛋白质粒真核表达载体pGenesil中,构建重组载体pGenesil-PLCε。重组载体转染T24细胞后,RT-PCR和Western blot检测PLCE基因和蛋白表达;以侵袭小室、明胶酶谱分析及免疫组化检测细胞侵袭能力。结果成功构建了针对PLCE基因的shRNA表达载体。两个阳性重组质粒P1和P2转染膀胱癌细胞T24,P1和P2组目的基因/内参照吸光度比值较空载HK-A组明显降低(P<0.01);P1和P2组目的蛋白/内参照吸光度比值较空载HK-A组明显降低(P<0.01)。细胞侵袭实验中阳性质粒P1和P2转染组穿膜细胞数也明显低于空白对照组和阴性质粒HK-A转染组(P<0.01);转染P1、P2均使细胞分泌MMP-2、MMP-9明显低于空白对照和HK-A转染组(P<0.01)。结论阻断PLCE基因能有效抑制膀胱癌T24细胞系中PLCE基因和蛋白表达,并对T24细胞的侵袭转移能力具有一定的抑制作用。更多还原

【Abstract】 Objective To construct a shRNA expression plasmid against gene PLCE and to observe the inhibition of PLCE gene expression in bladder cancer cell line T24.Methods pGenesil-PLCε plasmids were constructed and inserted into T24 cells,then RT-PCR,Western blot analysis was taken to know about inhibition of PLCE gene expression after the transfection of plasmids.Invasive power of T24 was measured before and after transfection by the membrane invasion culture system(Transwell chamber),gelatin enzymography and immunochemistry of cells.Results The two recombinant plasmids were constructed successfully,in P1 and P2 groups the absorbance ratio of PLCE mRNA to inner reference was lower than the HK-A group(P<0.01),and the absorbance ratio of PLCE protein to inner reference in P1 and P2 groups was lower than the HK-A group(P<0.01).The invasion number of T24 transfected with P1 and P2 are both lower than the the group of HK-A and group without transfection(P<0.01);MMP-2,MMP-9 had lower expression in gelatin enzymo-graphy assay and imunocytochemistry of cells than the groups HK-A and control(P<0.01).Conclusion RNAi of PLCE might decrease invasive power of bladder cancer cells so as to inhibit the development of tumor.更多还原