病毒介导的P53低表达HepG2细胞株的建立

Establishment of retrovirus-mediated P53 low expression HepG2 cell line

目的构建干扰P53的逆转录病毒载体,建立稳定低表达P53的HepG2细胞株。方法合成干扰P53基因的寡核苷酸序列插入pMSCV-hyg-U6质粒,转化受体菌DH5α,经酶切测序鉴定后瞬时转染HepG2细胞,通过半定量RT-PCR方法检测P53 mRNA表达水平评估干扰效果;选择干扰效果最好的重组质粒转染PT67细胞进行病毒包装,获得逆转录病毒颗粒感染HepG2细胞,通过Hygromycin筛选获得多个细胞克隆,Western blot检测P53蛋白的表达情况;选择P53表达水平最低的细胞克隆。通过5-氟尿嘧啶(5-FU)诱导凋亡实验,检测P53低表达的HepG2细胞p53通路介导的细胞凋亡情况。结果经酶切和测序验证成功构建重组病毒载体;瞬时转染HepG2细胞后P53 mRNA表达下调;病毒颗粒感染HepG2细胞后P53的mRNA、蛋白表达下调;用5-FU处理后P53低表达HepG2细胞凋亡水平明显低于对照。结论成功构建了干扰P53的逆转录病毒载体,建立了P53低表达HepG2细胞株,明显阻断了P53通路依赖的细胞凋亡。

In this study, we aimed to establish HepG2 cell line with stably low expression of P53 by using virus-mediated RNA interference technology. P53 siRNA retrovirus expression vectors were constructed with pMSCV-hyg-U6, and then transfected into PT67 package cells. Virus suspension was collected to infect HepG2 cell lines. RT-PCR was used to detect the expression level of P53 mRNA and Western blot was used to analyze the protein level of P53. HepG2 cell line with stably low expression of P53 was treated with 5-FU, and flow cytometry were used to detected cell apoptosis. We found that the expression level of P53 mRNA was down-regulated after HepG2 cells were transient transfected with the recombinant plasmid. The expression of P53 protein was down-regulated after HepG2 cells were infected with the recombinant retrovirus. Apoptosis level of HepG2 cell line with stably low expression of P53 was significantly lower than the control. In our study, the RNAi retroviral vector targeting to P53 was successfully constructed and HepG2 cell line with stably low expression of P53 were established, in which the P53 pathway-dependent apoptosis was significantly blocked. Thus this study would set a basis for evaluating the role of P53 pathway in the pathologic processes of infection and tumor.