脱氧雪腐镰刀菌烯醇对PC12细胞凋亡的影响

Effect of deoxynivalenol on apoptosis of PC12 cells

[目的]本试验旨在研究脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)诱导PC12细胞凋亡及对相关基因Bcl-2、Bax、Bid mRNA和蛋白cleaved-Caspase 3、cleaved-Caspase 9表达水平的影响。[方法]用不同质量浓度的DON(0、125、250、500、1 000和2 000 ng·m L^(-1))对PC12细胞染毒24 h,采用透射电镜观察、流式细胞术、荧光定量PCR、Western-blot等方法,研究DON对PC12细胞的超微结构、凋亡率、凋亡相关基因及蛋白表达的影响。[结果]不同浓度的DON均可诱导细胞凋亡,各试验组细胞凋亡率均极显著高于对照组(P<0.01),并随着染毒剂量的增加而升高,在1 000 ng·m L^(-1)时达到峰值;与对照组相比,各试验组Bax mRNA表达水平和Bax/Bcl-2值均极显著增加(P<0.01),Bcl-2 mRNA表达水平在DON浓度超过500 ng·m L^(-1)时极显著下降(P<0.01),而Bid mRNA表达水平在DON浓度超过250 ng·m L^(-1)时极显著下降(P<0.01);Western-blot检测结果显示,各试验组cleaved-Caspase 9蛋白表达量均极显著高于对照组(P<0.01),而cleaved-Caspase 3蛋白表达量在DON浓度超过500 ng·m L^(-1)时极显著增加。[结论]DON能够诱导PC12细胞发生凋亡,凋亡相关基因Bcl-2、Bax、Bid及蛋白Caspase3、Caspase9等参与了DON诱导PC12细胞凋亡的调控。

[Objectives]The experiment aimed at the effect of DON on Bcl-2,Bax,Bid mRNA and the cleaved-Caspase 3,cleavedCaspase 9 protein in PC12 cells. [Methods]PC12 cells were exposed to deoxynivalenol（ DON） at different concentrations（ 0,125,250,500,1 000,2 000 ng·m L^-1） for 24 h. The changes of cell ultrastructure,cell apoptosis ratio,apoptosis-related genes expression and apoptosis-related protein expression were determined by transmission electron microscopy,flow cytometry,q PCR and Westernblot,respectively. [Results]Different concentrations of DON could induce cell apoptosis,compared with control group,the cell apoptosis ratio in experimental groups significantly increased（ P0.01）,and the changes with the increase of DON concentrations,peak at1 000 ng·m L^-1. Compared with control group,the Bax mRNA and Bax/Bcl-2 ratio was significantly increased（ P 0.01）,the Bcl-2mRNA was significantly decreased（ P0.01） when the concentration of DON above 500 ng·m L^-1,and the Bid mRNA was significantly decreased（ P0.01） when the concentration of DON above 250 ng·m L^-1. Western-blot results showed that the cleaved-Caspase 9 in experimental groups were significantly increased（ P0.01）,however,the cleaved-Caspase3 significantly increased（ P0.01） when the concentration of DON was above 500 ng·m L^-1. [Conclusions]DON could induce PC12 cell apoptosis,and Bcl-2,Bax,Bid,Caspase3 and Caspase 9 participated in the regulation of cell apoptosis.