摘要
为探讨玉米赤霉烯酮(ZEA)的雄性生殖毒性及其机理,本试验研究了低质量浓度ZEA暴露对小鼠睾丸间质细胞株TM3细胞增殖及核内γH2AX荧光焦点产生的影响。以TM3细胞为材料,加入不同质量浓度的ZEA,设0,5,10,20μg/L ZEA染毒组,培养72h后,应用噻唑蓝比色法检测TM3细胞增殖活力,观察细胞周期分布及其调控蛋白Cyclin D1、CDK4表达情况,同时应用免疫荧光、蛋白免疫印迹法检测TM3细胞中γH2AX含量的变化。结果显示,染毒72h后,低质量浓度的ZEA对TM3细胞的生长具有明显的促进作用,检测细胞周期发现10μg/L ZEA染毒组G1/G0期细胞分布百分比显著下降(P<0.05),S期细胞分布百分比显著升高(P<0.05),20μg/L ZEA染毒组G1/G0期细胞分布百分比极显著下降(P<0.01),S期细胞分布百分比显著升高(P<0.05);通过免疫荧光发现,5μg/L ZEA染毒组中部分细胞开始出现1至2个荧光焦点,与对照组相比,10μg/L ZEA染毒组和20μg/L ZEA染毒组出现γH2AX荧光焦点的细胞数量明显增多,荧光焦点数呈极显著升高(P<0.01);同时通过蛋白免疫印迹法发现,ZEA能够促进γH2AX蛋白表达量的增加,对细胞周期调控相关蛋白Cyclin D1、CDK4的表达也有明显的促进作用。低质量浓度的ZEA有促进细胞增殖的作用,并可诱发TM3细胞的DNA双链断裂,呈剂量-效应关系。
In order to explain zearalenone-induced male reproductive toxicity and its mechanism,the effect of low quality concentration of zearalenone on proliferation, γH2AX fluorescent focus forma tion and its protein expression were investigated in TM3 cells（mice Leydig cell line）. TM3 cells were taken as materials, and then treated with various quality concentrations（0,5,10 and 20 μg/L） of ZEA for 72 h. Cell viability was determined by MTT assay. Then cell cycle,Cyclin D1 and CDK4 protein were observed. γH2AX changes were analyzed by immunofluorescence, Western blot. The results showed that,after exposure to zearalenone for 72 h,low quality concentration of ZEA could obviously promote the growth of TM3 cells. The detection of cell Cycle discovered that the percent- age of cell distribution decreased obviously in G1/G0 phase and increased significantly in S phase with 10,20 μg/L of ZEA（P〈0.05）. Immunofluorescence showed that a small part of the cell with 5 μg/L of ZEA began to display 1 to 2 fluorescent focus. Compared with the control group, the number of cells with 7H2AX fluorescent focus and the number of fluorescent focus with 10,20 γg/L of ZEA increased significantly（P〈0.01）. Western blot assay found that ZEA could raise the expression of 7H2AX,Cyclin D1,CDK4 protein. The results indicated that low concentration of ZEA could promote the growth of TM3 cells and induced DNA double-strand breaks in dose-de- pendent manners.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2017年第6期1167-1172,共6页
Chinese Journal of Veterinary Science
基金
江苏高校优势学科建设工程资助项目(PAPD)
江苏省普通高校自然科学基金资助项目(08KJD230002)
