p100^(Rb)和p80^(Rb)过表达对细胞周期和凋亡的影响  

Effects of p100^(Rb)and p80^(Rb)overexpression on cell cycle and apoptosis of HEK 293T cells

作　　者：李强 李彩红 周恒 乔兵 刘建军 范礼斌 Li Qiang;Li Caihong;Zhou Heng;Qiao Bing;Liu Jianjun;Fan Libin(Dept of Biology,School of Life Sciences,Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学生命科学学院生物系,合肥230032

出　　处：《安徽医科大学学报》2022年第9期1350-1355,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:11040606M170);安徽医科大学双肩挑干部学术恢复科研专项(编号:0112011109)。

摘　　要：目的探究视网膜母细胞瘤蛋白(Rb)截短体p100^(Rb)和p80^(Rb)对细胞周期、凋亡的影响。方法构建pcDNA3.1-p100^(Rb)-FLAG、pcDNA3.1-p80^(Rb)-FLAG真核表达质粒;分别转染至U2OS细胞中,进行免疫荧光制片,检测p100^(Rb)和p80^(Rb)细胞定位情况;将pcDNA3.1-RB1-FLAG、p100^(Rb)和p80^(Rb)分别转染至HEK 293T细胞中,提取总蛋白进行免疫印迹,检测Rb、p100^(Rb)和p80^(Rb)的过表达;过表达Rb、p100^(Rb)和p80^(Rb)后利用流式细胞术检测细胞周期、凋亡。结果成功构建pcDNA3.1-p100^(Rb)-FLAG和pcDNA3.1-p80^(Rb)-FLAG真核表达质粒;在U2OS细胞中p100^(Rb)主要定位在细胞核,p80^(Rb)主要定位在细胞质;Rb、p100^(Rb)和p80^(Rb)在HEK 293T细胞中可过表达;过表达Rb、p80^(Rb)和p100^(Rb)后,与对照组比较,过表达的Rb实验组和p100^(Rb)实验组细胞周期G 1期百分比升高,各实验组细胞的凋亡率均显著降低(均P<0.05)。结论过表达p100^(Rb)可抑制HEK 293T细胞凋亡且阻滞细胞周期;过表达p80^(Rb)可抑制HEK 293T细胞凋亡但对细胞周期无影响。Objective To investigate the effects of p100^(Rb)and p80^(Rb)truncated forms of retinoblastoma protein(Rb)on HEK 293T cell cycle and apoptosis.Methods The eukaryotic expression plasmid pcDNA3.1-p100^(Rb)-FLAG and pcDNA3.1-p80^(Rb)-FLAG were constructed,plasmid was transfected into U2OS cells and immunofluorescence was performed to define the localization of p100^(Rb)and p80^(Rb)in cells.Plasmid was also transfected into HEK 293T cells,and the total protein was extracted for Western blotting to detect the protein expression level.After overexpression of Rb,p100^(Rb)and p80^(Rb)in HEK 293T cells,cell cycle and apoptosis were measured by flow cytometry.Results The eukaryotic expression plasmid pcDNA3.1-p100^(Rb)-FLAG and pcDNA3.1-p80^(Rb)-FLAG were constructed successfully.p100^(Rb)was mainly located in the nucleus while p80^(Rb)was mainly located in the cytoplasm,in U2OS cells.Rb,p100^(Rb)and p80^(Rb)could be overexpressed in HEK 293T cells.Following overexpression of Rb,p100^(Rb)in HEK 293T cells,the percentage of G 1 phase of cell cycle significantly increased compared with the control group,and p100^(Rb)and p80^(Rb)inhibited apoptosis compared with control group(P<0.05).Conclusion Overexpression of p100rb can inhibit the apoptosis of HEK 293T cells and block the cell cycle.Overexpression of p80rb can inhibit the apoptosis of HEK 293T cells,but has no effect on cell cycle.

关 键 词：视网膜母细胞瘤蛋白 过表达 细胞周期 凋亡 

分 类 号：R34[医药卫生—基础医学] Q28[生物学—细胞生物学]