百草枯对人胚胎神经祖细胞自噬作用的影响

The effect of paraquat on autophagy in human embryonic neural progenitor cells

目的通过观察百草枯（PQ）染毒后人胚胎神经祖细胞超微结构变化以及自噬相关基因表达情况，探讨PQ对人胚胎神经祖细胞自噬作用的影响。方法以永生化的人胚胎神经干细胞系（ReNcell CX细胞）为研究对象，设立1．00、10．00、100．00μmol／LPQ染毒组和对照组（0．00μmol／L）。采用CCK8法测定PQ染毒对ReNcell细胞活力的影响，透射电镜观察细胞超微结构变化；实时荧光定量一聚合酶链反应技术检测自噬相关基因mTOR、Beclinl、Atg5、Atg7、LC3、Atg12的mRNA表达情况以及凋亡相关基因Bc-2和Bax的mRNA表达情况。结果与对照组比较，100．00μmol／L染毒组人胚胎神经干细胞活力明显下降，差异有统计学意义（P〈0．01）。1．00μmol／L染毒组可观察到自噬小体，但无细胞凋亡改变，而10．00μmol／L染毒组可同时可观察到自噬小体和细胞凋亡改变。自噬作用正向调节基因LC3、Atg12、Atg5、beclin1、Atg7 mRNA表达水平随PQ浓度的增加，呈现先增加后降低的趋势。1．00μmol/L染毒组beclinl mRNA表达水平达到峰值；10．00μmol／L染毒组LC3、Atg12、Atg5、Atg7 mRNA表达水平达到峰值。而自噬作用负调节基因mTOR mRNA表达水平随PQ浓度的增加呈现先下降后上升的趋势，与对照组比较1．00μmol／L染毒组mTOR mRNA表达水平明显下降，差异有统计学意义（P〈0．01）：100．00μmol／L1染毒组mTOR mRNA表达水平明显增加，差异有统计学意义（P〈0．01）。与对照组比较，10．00μmol／L染毒组促凋亡基因Bax mRNA表达水平明显下降，差异有统计学意义（P〈0．01），100．00μmol/L染毒组Bax mRNA表达水平明显增加，差异有统计学意义（P〈0.01）。1．00、10．00、100．00μmol／L1染毒组抗凋亡基因Bcl-2 mRNA表达水平均明显低于对照组，但在10．00μmol／L染毒组Bcl-2 mRNA表达水平最低，差异有统计学意（P〈0．01）。结论在PQ染毒浓度较低时激活了自噬作用，自噬作用发挥神经保护的作用；PQ染毒浓度较高时，自噬作用被抑制并诱导细胞凋亡增加。

Objective To explore the effect of paraquat （PQ） on autophagy in human embryonic neural progenitor cells. Methods Using ReNcell CX cell model .After treatment with various concentration （0.00, 1.00, 10.00 and 100.00 μmol/L） of PQ, CCK8 assay was used to detect the cell viability, the transmission electron microscope was used to observe the the cell ukrastructure, the real-time polymerase chain reaction（RT- PCR） was adopted to detect mRNA expression of autophagy related genes which including LC3, Atgl2, AtgS, beclinl, Atg7 and roTOR and apoptosis related genes Bax and Bcl-2. Results The cell viability was significantly inhibited after administered with 100.00 μmol/L of PQ （P〈0.01）. The mRNA expression of Beclinl was significantly up-regulated and the emergency of autophagosome were observed at the concentration of 1.00 μmol/L group, while mild cell apoptosis, significantly up-regulated Atg5, Atg8, Atg7 and Atgl2 mRNA expression as well as down-regulated expression of mTOR and Bax were detected at the 10.00 μmol/L of PQ group, howere, the obvious apoptosis and the up-regulated mRNA expression of mTOR and Bax were observed at the 100.00 μmol/L of PQ group, the mRNA expression of Bcl-2 were all down-regulated after administered with 1.00, 10.00 and 100.00 μmol/L of PQ and reached the lowest level at the concentration of 10.00 μmol/L. Conclusion PQ can induced autophagy at the low concentration in ReNeell CX cell and autophagy might serve as a protective mechanism to ameliorate PQ-induced cytotoxie effects but apoptosis will be induced at the 100 μmol/L concentration.