Microcystin-LR对体外牛子宫内膜上皮细胞增殖和凋亡的影响

Effects of microcystin-LR on proliferation and apoptosis of bovine endo metrial epithelial cells in vitro

使用Microcystin-LR体外处理牛子宫内膜上皮细胞,探究其对细胞活率影响及分子调控机制,以期为提高牛繁殖效率提供理论基础和实践依据。试验运用CCK-8检测MC-LR处理后细胞存活率;流式细胞术检测细胞周期变化和细胞凋亡率;Annexin V-FITC试剂盒检测细胞凋亡;实时荧光定量检测Pi3k、Akt、mTOR、Cyt-c、Caspase-3、Caspase-9和p53基因mRNA表达量。Western blot检测Pi3k、Akt、Caspase-3和Caspase-9蛋白表达量。结果表明,不同浓度和时间处理下细胞存活率出现升高和降低两种相反情况,根据CCK-8检测结果,选择12 h,100μg·L^(-1)和24 h,160μg·L^(-1)两种处理方案开展后续试验。在12 h,100μg·L^(-1)条件下,Pi3k、Akt、mTOR的mRNA表达量升高(P<0.01),Cyt-c、Caspase-3和Caspase-9基因mRNA表达量下降(P<0.05),Pi3k和Akt蛋白表达量升高(P<0.05),Caspase-3和Caspase-9蛋白表达量降低(P<0.05)。处于G0/G1期细胞数量减少(P<0.01),处于G2/M和S期细胞数量增加(P<0.01);p53基因mRNA表达量升高(P<0.05)。在24 h,160μg·L^(-1)处理条件下,Caspase-3、Caspase-9、Bax/Bcl2基因mRNA表达量升高(P<0.05),Caspase-3和Caspase-9蛋白表达量升高(P<0.05)。研究表明,100μg·L^(-1)MC-LR处理细胞12 h,细胞通过增强Pi3k-Akt-mTOR信号通路,抑制线粒体凋亡通路促进细胞增殖,同时导致处于G1期细胞数量减少、G2/M期和S期细胞数量增加。160μg·L^(-1)MC-LR处理细胞24 h,细胞通过活化线粒体凋亡通路发生凋亡。

Treating bovine endometrial epithelial cells with Microcystin-LR in vitro explore its effects on cell viability and the mechanism of molecular regulation,in order to provide theoretical and practical basis for improving the efficiency of bovine reproduction.CCK 8 was used to detect cell survival rate after treatment;flow cytometry was used to detect cell cycle changes and cell apoptosis rate;Annexin V-FITC kit was used to detect cell apoptosis;real-time fluorescence quantitative detection of Pi3k,Akt,mTOR,Cyt-c,Caspase-3,Caspase-9 and p53 gene mRNA expression levels.Western blot was used to detect the protein expression of Pi3k,Akt,Caspase-3 and Caspase-9.The cell survival rate under different concentrations and time treatments showed two opposite results:increase and decrease.According to the CCK 8 test results,two treatment options of 12 h,100μg·L^(-1) and 24 h,160μg·L^(-1) were selected for follow-up experiments.At 12 h,100μg·L^(-1) treatment conditions,the mRNA expression of Pi3k,Akt,mTOR were increased(P<0.01),and the mRNA expression of Cyt-c,Caspase-3 and Caspase-9 were genes decreased(P<0.05).The expression of Pi3k and Akt protein were increased(P<0.05),and the expression of Caspase-3 and Caspase-9 protein were decreased(P<0.05).The number of cells in the G0/G1 phase decreased(P<0.01),and the number of cells in the G2/M and S phase was increased(P<0.01);the mRNA expression of p53 gene was increased(P<0.05).In 24 h,160μg·L^(-1) treatment conditions,Caspase-3,Caspase-9,Bax/Bcl2 gene mRNA were expression increased(P<0.05),Caspase-3 and Caspase-9 protein expression were increased(P<0.05).Cells were treated with 100μg·L^(-1) MC-LR for 12 h,cells promoted cell proliferation by enhancing Pi3k-Akt-mTOR signaling pathway and inhibiting mitochondrial apoptosis pathway,reducing the number of cells in G1 phase,and increasing cells in G2/M and S phases.After treating the cells with 160μg·L^(-1) MC-LR for 24 h,the cells undergo apoptosis by activating the mitochondrial apoptosis pathway.