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目的:构建shRNA体内表达载体pGeneClipTM-shRNA,研究抑制Bmi-1基因表达对膀胱癌5637细胞凋亡的影响。方法:构建靶向Bmi-1的shRNA真核表达质粒pGe-neClipTM-B1、pGeneClipTM-B2以及pGene-ClipTM-B-neg(阴性对照质粒),采用脂质体转染试剂转染膀胱癌5637细胞,采用RT-PCR、蛋白质印迹法技术检测转染前后5637细胞中Bmi-1基因表达的变化;利用MTT法检测表达质粒对5637细胞增殖的影响;并利用流式法和蛋白质印迹法技术检测表达质粒对5637细胞凋亡的作用及影响机制。结果:经酶切、测序鉴定,重组质粒均在1 200 bp左右出现酶切条带,符合设计要求。RT-PCR和蛋白质印迹法检测结果显示,5637细胞经小RNA干扰后Bmi-1的mRNA和蛋白质表达水平明显降低,P<0.05。与阴性对照组和未转染组相比,转染Bmi-1 shRNA组细胞增殖明显受到抑制,P<0.05。转染Bmi-1 shRNA组细胞凋亡率分别为19.95%和27.27%,与未转染组7.23%和阴性对照组6.78%相比,转染Bmi-1 shRNA组细胞凋亡率明显增加,P<0.05。结论:减少膀胱癌5637细胞Bmi-1基因的表达可抑制5637细胞增殖并促进细胞凋亡。
OBJECTIVE: To construct shRNA expression vector pGeneClipTM-shRNA to study the effect of inhibiting the expression of Bmi-1 gene on the apoptosis of bladder cancer 5637 cells. Methods: The eukaryotic expression plasmids pGe-neClipTM-B1, pGeneClipTM-B2 and pGene-ClipTM-B-neg (negative control plasmids) targeting Bmi-1 were constructed and transfected into bladder cancer 5637 cells The expression of Bmi-1 gene was detected by RT-PCR and Western blotting in 5637 cells before and after transfection. The effect of expression plasmid on the proliferation of 5637 cells was detected by MTT assay. Flow cytometry and Western blotting were used to detect the expression of Bmi- Effect of Plasmid on Apoptosis of 5637 Cells and Its Mechanism. Results: After restriction enzyme digestion and sequencing, the recombinant plasmids showed digestion bands around 1 200 bp, which met the design requirements. The results of RT-PCR and Western blotting showed that the mRNA and protein expression of Bmi-1 in 5637 cells were significantly decreased after small RNA interference (P <0.05). Compared with negative control group and untransfected group, the proliferation of Bmi-1 shRNA transfected group was significantly inhibited (P <0.05). The apoptosis rate of transfected Bmi-1 shRNA group was 19.95% and 27.27%, respectively, compared with 7.23% in untransfected group and 6.78% in negative control group, the apoptosis rate of transfected Bmi-1 shRNA group was significantly increased, P <0.05. Conclusion: To reduce the expression of Bmi-1 gene in bladder cancer 5637 cells can inhibit the proliferation and promote the apoptosis of 5637 cells.