【摘要】 目的观察谷氨酰胺(Gln)对Hela细胞凋亡的影响,探讨该作用与蛋白激酶B(Akt)-Yes相关蛋白(YAP)信号转导通路的关系。方法用含不同浓度Gln(4、2、1、0.5、0.1、0mmol/L)的Dulbecco改良Eagle培养基(DMEM培养基)分别培养Hela细胞24h,应用细胞计数试剂盒-8(CCK-8)检测细胞活性。用含Gln4或0mmol/L或Gln 4mmol/L+Akt抑制剂LY294002 40μmol/L的DMEM培养基分别培养Hela细胞24h,采用Western印迹法检测B细胞淋巴瘤/白血病-2相关X蛋白(Bax)、磷酸化Akt(pAkt)、磷酸化YAP(pYAP)蛋白的表达。利用小干扰RNA(siRNA)抑制Hela细胞YAP的表达,用含Gln 4或0mmol/L的DMEM培养基分别培养正常细胞或YAP siRNA转染后的细胞24h,采用Western印迹法检测Bax蛋白的表达。结果以含4、2、1、0.5、0.1、0mmol/L Gln的DMEM培养基分别孵育Hela细胞24h后,Gln 1、0.5、0.1、0mmol/L组的细胞活性均显著低于Gln 4mmol/L组(P值分别<0.05、0.01)。以含Gln 4或0mmol/L的DMEM培养基孵育Hela细胞24h后,Gln 0mmol/L组的pAkt和pYAP水平较Gln 4mmol/L组显著降低(P值均<0.01),Bax蛋白表达显著增加(P<0.01)。当Akt抑制剂LY294002 40μmol/L与Gln 4mmol/L共同孵育细胞24h后,与Gln 4mmol/L组相比,pAkt和pYAP水平显著降低(P值均<0.01),而Bax蛋白表达显著增加(P<0.01)。正常或YAP siRNA转染的Hela细胞分别在含Gln 4或0mmol/L的DMEM培养基中培养24h后,与正常细胞Gln 4mmol/L组比较,正常细胞Gln 0mmol/L组Bax蛋白表达显著增加(P<0.01),而YAP siRNA转染后细胞Gln 0mmol/L组Bax蛋白表达则显著降低(P<0.05)。结论 Gln饥饿可诱导Hela细胞凋亡,其作用机制与Akt活性降低,进而导致YAP去磷酸化后入核诱导Bax表达增加有关。更多还原

【Abstract】 Objective To investigate the effect of glutamine on apoptosis of Hela cells,and to explore the relationship between this effect and the protein kinase B(Akt)-Yes-associated protein(YAP)signaling pathway.Methods Hela cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)with different concentrations of glutamine(4,2,1,0.5,0.1,0mmol/L)for 24hours,and the cell viability was measured by cell counting kit 8(CCK-8).Then Hela cells were cultured in DMEM with glutamine 4mmol/L,glutamine 0mmol/L or glutamine 4mmol/L+ Akt inhibitor LY294002 40μmol/L for another 24hours;Western blotting analysis was used to detect the expression of Bcl-2associated X protein(Bax),phosphorylated protein kinase B(pAkt)and phosphorylated Yes-associated protein(pYAP).YAP expression was suppressed by siRNA.Normal cells or YAP siRNA transfected cells were cultured in DMEM with glutamine 4or 0mmol/L for 24hours,and Bax expression was measured by Western blotting analysis.Results After 24hours incubation with different concentrations of glutamine,cell viability of glutamine 1,0.5,0.1,and 0mmol/L groups decreased significantly as compared with glutamine 4mmol/L group(P<0.05or 0.01).Compared with glutamine 4 mmol/L group,Akt and YAP phosphorylation levels was decreased and Bax expression was increased in glutamine 0 mmol/L group andglutamine 4mmol/L+LY294002 40μmol/L group(all P<0.01).Bax expression was increased in Gln 0mmol/L of normal cells after incubation for 24hours(P<0.01),while it was decreased in YAP siRNA transfected cells(P<0.05).Conclusion Glutamine starvation can induce apoptosis of Hela cells,and the possible mechanism is related to decreased Akt activity,leading to dephosphorylation YAP into the nuclear and eventually to increased expression of Bax.更多还原