PP2R1A逆转录病毒的构建及对细胞周期的影响

目的：构建重组PP2R1A基因的逆转录病毒感染HEKTER细胞，观察其定位，验证表达，研究过表达PP2R1A对细胞生长及周期的影响。方法：逆转录病毒载体pMIG-Flag-PP2R1A-IRES-GFP与Pcll0A1瞬时共转染293T细胞，收集病毒感染HEKTER细胞，在荧光显微镜下观察定位，标记荧光单克隆。挑取不同表达强度单克隆做western验证PP2R1A蛋白表达。运用流式细胞分析、体外创伤试验及生长曲线试验研究单克隆细胞的增殖及周期。结果：获得了过表达PP2R1A的单克隆细胞株，PP2R1A在细胞内广泛表达，结合western及细胞试验证实PP2R1A高表达阻滞细胞周期并减慢细胞生长。结论：PP2R1A是丝苏氨酸蛋白磷酸酶PP2A的结构A亚基的a亚型，在细胞内广泛表达。本文成功构建了表达PP2R1A的细胞株，研究发现PP2R1A高表达会影响细胞生长及细胞周期，减缓了细胞增殖。为进一步深入研究PP2R1A对PP2A全酶活性及功能、细胞转化的影响奠定了重要的实验基础。

Construction of retrovirus and the influnence on cell cycle of PP2R1A

To construct the recombinant PP2R1A （PP2A Aa subunit）-containing retrovirus,and infect HEKTER cells using the retrovirus expression system.Then Investigate the intracellular location and overexpression of PP2R1A,also the effect on cell multiplication and cell cycle.Methods：The retrovirus plasmid pMIG-Flag-PP2R1A-IRES-GFP was co-transfected into 293T cell with Pcll0Al,the virus were collected to infect HEKTER. The intraeellular location and selection of monoclonal cell lines were determined using the fluorescencemicroscope, and the overexpression of PP2R1A was detected by western blotting Flow cytometry, in vitro wound healing and cell growth curve assay were performed to investigate the effect on cell multiplication and cell cycle.Results＇The PP2R1A-overexpression monoclonal cell lines were selected,and PP2R1A was widely expressed in cell.The result of western and cell biology analysis showed that overexpression of PP2R1A inhibit the cell cycle and suppress the growth of cell. Conclusions：PP2R1A is the structural subunit Act of PP2A which is one of the serine-threonine phosphatases family, and is widely expressed in cell.Here we successfully obtained PP2R1A-overexpression monoclonal cell lines.The results showed that the overexpression of PP2R1A had influence on cell growth and cell cycle,also suppress the cell multiplication.These layed an important experimental foundation for the further study of PP2R1A and PP2A holoenzymes.