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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。
Objective To construct a retroviral vector that interfered with P53 and establish a HepG2 cell line stably expressing low P53. Methods The oligonucleotide sequence of P53 gene was inserted into pMSCV-hyg-U6 plasmid and transformed into recipient strain DH5α. The recombinant plasmid was transiently transfected into HepG2 cells after digestion and sequencing. The expression of P53 mRNA was detected by semi-quantitative RT-PCR Interfering effect; Select the best interference plasmid transfected PT67 cells for viral packaging, get retroviral particles infected HepG2 cells, obtained by Hygromycin screening of multiple cell clones, Western blot detection of P53 protein expression; select P53 expression level The lowest cell clone. Apoptosis was detected by 5-fluorouracil (5-FU), and p53 pathway-mediated apoptosis in P53-low expression HepG2 cells was detected. Results Recombinant virus vector was successfully constructed by restriction enzyme digestion and sequencing. The expression of P53 mRNA was down-regulated after HepG2 cells transient transfection. The mRNA and protein expression of P53 were down-regulated after HepG2 cells were infected with virus particles. Death level was significantly lower than the control. Conclusion The retroviral vector that interfered with P53 was successfully constructed and HepG2 cell line with low P53 expression was established, which obviously blocked the apoptosis of P53 pathway-dependent cells.