FAM172A蛋白对人胚胎肾细胞凋亡及增殖的影响

目的 研究与糖尿病大血管病变有关的新蛋白FAM172A对人胚胎肾细胞(HEK293细胞)凋亡及增殖的影响.方法 以前期研究工作中所构建的pDrive-FAM172A质粒为模板,通过PCR方法扩增出人FAM172A基因编码框,将扩增产物亚克隆到PDC315真核表达载体,经酶切、测序鉴定,选择插入序列正确的PDC315-FAM172A质粒用脂质体2000转染HEK293细胞,同时用PDC315空质粒作为对照转染HEK293细胞.用噻唑蓝(XTT)法、生长曲线法观察过表达FAM172A基因对HEK293细胞增殖的影响.用碘化丙啶(PI)单染色法、膜联蛋白V/PI双染色法观察过表达FAM172A基因对HEK293细胞凋亡及细胞周期的影响.结果 成功构建PDC315-FAM172A真核表达载体,经酶切、测序证实插入序列正确.与PDC315质粒转染相比,XTT显示PDC315-FAM 172A质粒转染使细胞增殖增加约52%(A值为0.21±0.07比0.32±0.06,P<0.01);生长曲线显示PDC315-FAM172A质粒转染使HEK293细胞生长速度增快;PI单染色法显示PDC315-FAM172A质粒转染使细胞凋亡减少约38.5%(分别23.79%±1.36%比14.64%±0.95%,P<0.01),同时G0～G1期细胞明显减少(分别66.79%±1.73%比58.16%±0.75%,P<0.01)而S期细胞明显增加(分别22.62%±1.16%比33.56%±0.94%,P<0.01);膜联蛋白V/PI双染色法显示PDC315-FAM172A质粒转染使早期凋亡细胞减少约28%(13.63%±0.56%比9.79%±0.39%,P<0.01),晚期凋亡细胞减少约29%(7.70%±0.29%比5.43%±0.29%,P<0.01).结论 FAM172A蛋白促进HEK293细胞增殖、抑制凋亡、促使细胞进入S期,提示FAM172A蛋白的生理功能之一是参与细胞生长的调控,这为深入研究FAM172A蛋白的生理功能及在糖尿病大血管并发症中的作用提供了线索.

Effect of FAM172A protein on apoptosis and proliferation in HEK293 cells

Objective To study the effect of FAM172A protein related to diabetic macroangiopathy on apoptosis and proliferation in HEK293 cells.Methods The pDrive-FAM172A plasmid constructed in our previous study was used as a template to amplify human FAM172A open reading frame by a polymerase chain reaction.The resulting PCR products were subcloned into the eukaryotic expression vector PDC315 to construct recombinant PDC315-FAM172A plasmid.PDC315-FAM172A plasmid was identified by enzyme cleavage and sequencing analysis.HEK293 cells were transiently transfected respectively with appropriate PDC315 or PDC315-FAM172A plasmid by Lipofectamine 2000 according to the manufacturer's instruction.XTT assay and growth curve were used to observe the effect of over-expression of FAM172A gene on HEK293cell proliferation.PI and Annexin V/PI staining method were used to assess the effect of FAM172A gene on apoptosis and cell cycle of HEK293 cell.Results Eukaryotic expression vector PDC315-FAM172A was successfully constructed and identified by enzyme cleavage and sequencing analysis.Compared with PDC315plasmid transfection,the XTT assay showed that optical density (A) value increased by 52% when transfected with PDC315-FAM172A plasmid (0.21±0.07 vs 0.32±0.06,P < 0.01).Growth curve revealed that HEK293 cells transfected with PDC315-FAM172A plasmid proliferated faster than those transfected with PDC315 plasmid.PI staining showed that,as compared with PDC315 plasmid transfection,the apoptotic rate of HEK293 cells transfected with PDC315-FAM172A plasmid decreased by 38.5%(23.79±1.36 vs 14.64±0.95,P < 0.01),cell percentage of G0-G1 phases significantly decreased (66.79±1.73 vs 58.16 ±0.75,P <0.01) and cell percentage of S phases significantly increased (22.62 ±1.16 vs33.56 ±0.94,P <0.01).Annexin V/PI staining revealed that,as compared with PDC315 plasmid transfection,the percentage of early and advanced apoptotic cells decreased by 28% (13.63 ±0.56 vs 9.79 ±0.39,P <0.01) and 29% (7.70±0.29 vs 5.43±0.29,P < 0.01) respectively.Conclusion FAM172A protein promotes cell proliferation,inhibits cell apoptosis and facilitates S-phases entry.It indicates that FAM172A protein is involved in cell growth regulation.Our findings provide a clue for further study on its physiological functions and roles in diabetic macroangiopathy.