不同营养状态下柯萨奇病毒B3感染HeLa细胞后对mTOR及p70S6KmRNA的影响

Effect of HeLa cells infected with Coxsackie virus B3 on mTOR and p70S6K mRNA expression under different nutritional conditions

目的探讨不同营养状况下柯萨奇病毒B3（CVB3）感染HeLa细胞对mTOR信号通路的影响。方法采用非饥饿法和饥饿法2种方法培养HeLa细胞。（1）非饥饿法：HeLa细胞融合成40％～50％的单层细胞时，用含10g／L胎牛血清的细胞培养液换液培养24h后，病毒组用100倍半数感染量（100TCID50）的病毒干预，而对照组用病毒维持液（含胎牛血清2g／L细胞培养液）培养；（2）“饥饿法”：待HeLa细胞融合成40％～50％的单层细胞时，用不含胎牛血清的培养基换液培养24h后，病毒组用100TCID50的病毒干预，而对照组用病毒维持液（含胎牛血清2嚣／L的细胞培养液）培养。采用倒置显微镜观察细胞形态学变化，用实时荧光定量PCR检测3、6、9、12、24h不同时间点饥饿法及非饥饿法不同营养状态下HeLa细胞roTOR、p70S6KmRNA表达。结果非饥饿法：病毒组仅在12h及24hmTOR、p70S6KmRNA表达显著低于对照组（P均〈0．05）；饥饿法：病毒组各时间点均显著低于对照组（P均〈0．05）；饥饿法病毒组和对照组Hela细胞roTOR、p70S6KmRNA表达均高于非饥饿法，mTORmRNA差异均有统计学意义（P均〈0．05），而p70S6KmRNA差异均无统计学意义（P均〉0．05）。结论CVB3使HeLa细胞roTOR—PTOS6K信号通路表达下凋。

Objective To explore the effect of HeLa ceils infected with Coxsackie virus B3 （CVB3） on the changes of mTOR signal pathway under different nutritional conditions. Methods The HeLa cells were cultured under conventional culture and serum starvation culture. （1） For the conventional method,the medium with 10 g/L fetal bo- vine serum was added for 24 h after the Hela ceils were fused into 40% to 50% ,and the medium was changed on the next day,then the virus group was infected with CVB3 of 50% tissue culture infective dose （TCIDs0）. However, the control group was cultured by 2 g/L fetal bovine serum. （2） For the serum starvation method,HeLa cells were cultured with the medium without fetal bovine serum for 24 h. Then the virus group was infected with CVB3 of TCIDs0. The cells in control group were cultured by 2 g/L fetal bovine serum. Cell morphology changes were observed by inverted micro- scope, and the expressions of the mTOR,p70S6K mRNA were detected with Real-time PCR at 3 h,6 h,9 h, 12 h,24 h respectively in both conventional culture and serum starvation groups. Results The expressions of mTOR and pTOS6K mRNA were lower in the virus group than those in control group at 12 h and the 24 h （all P 〈0.05） in the conventio- nal culture group. And the expressions of mTOR and pTOS6K mRNA in the virus group were lower than those in the control group at every time points （ all P 〈 0.05 ） in serum starvation group. The expressions of mTOR and p70S6K mRNA in group with serum starvation virus and the control groups were higher than those in conventional culture group in all time points, but only the expressions of roTOR mRNA were significantly different between the 2 groups （ all P 〈 0. 05 ） , however, the expressions of p70S6K mRNA had no significant difference between the 2 groups （all P 〉 0.05 ）. Conclusion CVB3 may be able to down-regulate the expressions of mTOR and p70S6K mRNA.