重金属Cd对大鼠睾丸间质瘤细胞R2C孕酮合成的影响

Effect of Heavy Metal Cd on Progesterone Biosynthesis of R2C Cells

摘要:

探讨了Cd对体外大鼠睾丸间质瘤细胞R2C合成孕酮通路的影响，用MTT（噻唑蓝）比色法检测CdSO4对R2C细胞生长抑制率的影响，通过数据分析，确定CdSO4作用于R2C细胞的IC25、IC50、IC75浓度值，用IC25、IC50、IC75 3种浓度的CdSO4溶液培养R2C细胞，再采用碘标放射免疫法、流式细胞分析仪、ROS检测试剂盒和Western Blot法测定IC25、IC50、IC75对孕酮合成、线粒体膜电位、ROS和孕酮合成相关蛋白StAR、CYP11A1的影响。结果表明，CdSO4可以显著抑制R2C细胞的增殖，且CdSO4作用于R2C细胞的IC25、IC50、IC75浓度值分别为（24.93±0.023 5）、（44.80±0.047 9）、（80.51±0.035 7） μmol/L；IC25、IC50、IC75 3种浓度均能影响R2C孕酮合成；CdSO4可以增加R2C细胞ROS的产生和线粒体膜电位的损伤比率；CdSO4可以下调StAR、CYP11A1蛋白的表达，且对StAR蛋白的影响更为显著。重金属Cd可刺激R2C产生过量的ROS、下调StAR、CYP11A1蛋白的表达，进而影响线粒体的正常作用以及阻碍孕酮合成通路，最终影响孕酮的合成。

Abstract:

The effect of heavy metal Cd on progesterone synthesis pathways of R2C Cells were explored in this research，MTT assay was used to select different concentrations（IC25，IC50 and IC75） of CdSO4. And then R2C cells were stimulated by the three concentrations（IC25，IC50 and IC75） of CdSO4. Radioimmunoassay（RIA） was used to detect the progesterone biosynthesis. Flow cytometry（FCM） assay was used to detect the change in mitochondrial membrane potential（MMP）. The reactive oxygen species（ROS） were detected by ROS kit. The expression levels of StAR and CYP11A1 proteins were measured by Western Blot. The results showed that CdSO4 could inhibit the proliferation of R2C cells，and concentrations of IC25，IC50 and IC75 were（24.93±0.023 5）、（44.80±0.047 9）、（80.51±0.035 7）μmol/L. All of the three concentrations of CdSO4（IC25，IC50 and IC75） could inhibit progesterone biosynthesis，improve the production of ROS and increase the damage ratio of mitochondrial membrane. CdSO4 could also decrease the expression of StAR and CYP11A1. Conclsion：heavy metal Cd could stimulate R2C to produce excessive reactive oxygen species，decrease the level of StAR and CYP11A1，and in turn affect the normal function of mitochondria，ultimately affect the synthesis of progesterone.