RMP基因的表达及其对肝癌细胞凋亡的影响

目的制备RPB5调节蛋白（RMP）抗体并研究RMP在60Coγ射线诱导的SMMC-7721肝癌细胞凋亡过程中的表达及功能。方法 RT-PCR检测HT-29、A549、Hep-2、SGC-7901、293T、HeLa、WI-38和SMMC-7721细胞株中RMP的表达;从质粒中PCR扩增得到人RMP基因并克隆至原核表达载体中,诱导表达后经亲和层析纯化获得融合蛋白GST-RMP/D1,以该蛋白作为抗原免疫家兔制备多克隆抗体;肝癌细胞SMMC-TT21、PFlaG-CMV4-SMMC-7721、PFlaG-CMV4-RMP-SMMC-7721经6 Gy 60 Coγ射线照射后,流式细胞术检测肝癌细胞凋亡的变化。结果RMP在以上细胞系中均有表达,但程度不同;SDS-PAGE鉴定示纯化蛋白为目的蛋白人RMP片段。Western blot结果显示,制备的多抗可与RMP蛋白特异性结合;肝癌细胞株经6 Gy 60Coγ射线照射后,流式细胞术结果表明RMP使肝癌细胞SMMC-7721的凋亡减少。结论 RMP在不同细胞株中均有表达且能减少肝癌细胞SMMC-7727的凋亡,为RMP用于肝癌的基因治疗奠定了基础。

Expression of RPB5-mediating Protein and Effects on Apoptosis of Hepatoma Cells

Objective To prepare RPB5-mediating protein（RMP） polyclonal antibody and to investigate the expression and effect of RMP on apoptosis in human hepatoma cells line SMMC-7721 induced by 60Co γ-ray.Methods RMP mRNA expression in HT-29,A549,Hep-2,SGC-7901,293T,HeLa,WI-38 and SMMC-7721 cells was detected by RT-PCR.PCR from plasmid for RMP gene was cloned into prokar-yotic expression vector,the GST-RMP fusion protein was induced,purified with glutathione-sepharose beads,then rabbits were immunized with the purified protein RMP for obtaining the antiserum.Exposing to 60Co γ-ray irradiation at a dose of 6 Gy,flow cytometry was applied to measure the effect of RMP on cell apoptosis in SMMC-7721.Results RMP was expressed on cells of these cell lines.The SDS-PAGE showed target protein was the interest protein RMP.Preparation of polyclonal antibody binding with RMP protein specifically by Western blot.Exposing to the irradiation of the 60Co γ-ray at a dose of 6 Gy,flow cytometry was different applied to showed RMP reducing the apoptosis of SMMC-7721.Conclusion RMP expressed on cells of cell lines and reduces the apoptosis of SMMC-7721,which provides a way in liver cancer gene therapy.