低浓度玉米赤霉烯酮对TM3细胞增殖及γH2AX焦点形成的影响

发布时间：2018-12-27 17:40

【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(P0.05),S期细胞分布百分比显著升高(P0.05),20μg/L ZEA染毒组G1/G0期细胞分布百分比极显著下降(P0.01),S期细胞分布百分比显著升高(P0.05);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(P0.01);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。
[Abstract]:In order to study the male reproductive toxicity and mechanism of zeatin scinalenone (ZEA), the effects of low concentration ZEA exposure on the proliferation of TM3 cells and the production of 纬 H2AX fluorescence focus in nucleus of mouse testis stromal cell line were studied. TM3 cells were used as materials and ZEA, of different concentration was added to the control group. After 72 hours of culture, the proliferative activity of TM3 cells was detected by thiazolyl blue colorimetry, and the cell cycle distribution and the expression of Cyclin D1 + CDK4 were observed. At the same time, immunofluorescence and Western blot were used to detect the change of 纬 H2AX content in TM3 cells. The results showed that low concentration of ZEA significantly promoted the growth of TM3 cells 72 h after exposure, and the percentage of cell distribution in G1/G0 phase of 10 渭 g / L ZEA group decreased significantly (P0.05). The percentage of cell distribution in S phase was significantly increased (P0.05), and in 20 渭 g / L ZEA group, the percentage of cell distribution in G1/G0 phase was significantly decreased (P0.01), S phase cell distribution percentage was significantly increased (P0.05); It was found by immunofluorescence that some cells in 5 渭 g / L ZEA group began to present 1 or 2 fluorescent focal points. Compared with the control group, the number of cells exposed to 10 渭 g / L ZEA and 20 渭 g / L ZEA was significantly higher than that of the control group, and the number of cells exposed to 10 渭 g / L ZEA and 20 渭 g / L ZEA was significantly higher than that of the control group. The number of fluorescence focal points increased significantly (P0.01). At the same time, it was found by Western blotting that ZEA could promote the expression of 纬 H2AX protein and the expression of Cyclin D1 / CDK4, which was related to cell cycle regulation. Low concentration of ZEA could promote cell proliferation and induce DNA double strand breaks in TM3 cells in a dose-effect manner.
【作者单位】：扬州大学兽医学院;江苏省动物重要疫病与人兽共患病防控协同创新中心;
【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)
【分类号】：S859.8

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