| 付鹤玲李靓云李蕾李建民△.PP2R1A 逆转录病毒的构建及对细胞周期的影响[J].,2011,11(10):1869-1872 |
| PP2R1A 逆转录病毒的构建及对细胞周期的影响 |
| Construction of retrovirus and the influnence on cell cycle of PP2R1A |
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| DOI： |
| 中文关键词: PP2R1A 单克隆 亚细胞定位 细胞周期 |
| 英文关键词: PP2R1A Monoclonal cell line Intracellular location Cell cycle |
| 基金项目:国家自然科学基金资助项目（30971092） |
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| 中文摘要: |
| 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Pcl10A1 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。 |
| 英文摘要: |
| Objective: 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
Pcl10A1,the virus were collected to infect HEKTER. The intracellular 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 Aαof 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. |
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