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目的构建高效稳定的Bcl-2基因的RNA干扰慢病毒载体,检测Bcl-2的表达对细胞凋亡的影响。方法构建人Bcl-2慢病毒短发夹RNA(shRNA)干扰载体Lenti-H1 shRNA,转染HEK293T细胞,通过实时定量PCR及Western blot法检测Lenti-H1 shRNA的干扰效果。Lenti-H1 shRNA与4个包装质粒共同转染HEK293T细胞,包装成慢病毒后,分别感染ZR75-1和MCF-7乳腺癌细胞;嘌呤霉素筛选2周,收集细胞进行Western blot法检测Bcl-2蛋白的水平;采用流式细胞术检测乳腺癌细胞的凋亡情况。结果构建的Lenti-H1 Bcl-2 shRNA能够有效抑制Bcl-2蛋白的表达;流式细胞术结果显示,Bcl-2表达下调后明显促进细胞凋亡。结论慢病毒介导的Bcl-2蛋白下调能促进乳腺癌细胞的凋亡。
Objective To construct efficient and stable RNA interference lentiviral vector of Bcl-2 gene and detect the effect of Bcl-2 expression on apoptosis. Methods shRNA Lenti-H1 shRNA targeting human Bcl-2 lentivirus was constructed and transfected into HEK293T cells. The interference effect of Lenti-H1 shRNA was detected by real-time PCR and Western blot. Lenti-H1 shRNA and four packaging plasmids were co-transfected into HEK293T cells, which were packaged into lentivirus and infected respectively with ZR75-1 and MCF-7 breast cancer cells. Puromycin was screened for 2 weeks and cells were harvested for Western blotting to detect Bcl- 2 protein levels; using flow cytometry detection of breast cancer cell apoptosis. Results Lenti-H1 Bcl-2 shRNA could effectively inhibit the expression of Bcl-2 protein. The results of flow cytometry showed that Bcl-2 expression was significantly decreased after Bcl-2 shRNA treatment. Conclusion The down-regulation of lentivirus-mediated Bcl-2 protein can promote the apoptosis of breast cancer cells.