双酚A对体外大鼠睾丸支持细胞闭锁蛋白Occludin表达的影响

Effect of bisphenol A on expression of occludin of Sertoli cells in vitro rat

目的观察双酚A（BPA）对体外大鼠睾丸支持细胞增殖能力与闭锁蛋白Occludin（OCLN）表达的影响，探讨BPA对精子发生的损伤机制。方法体外分离培养雄性Wistar大鼠睾丸支持细胞，油红O染色鉴定。实验分为BPA染毒组（25laM、50uM和100IIM分别处理细胞24h）、溶剂对照组（无血清DMEM／F12＋DMSO＋细胞悬液）和空白对照组（无血清DMEM／F12）。CCK-8法测支持细胞增殖活性，WesternBlot法检测OCLN表达水平。结果分离培养大鼠睾丸支持细胞纯度〉90％。CCK-8实验结果显示：BPA对支持细胞增殖活性具有抑制作用，BPA浓度〉103uM时，存活率明显降低，细胞存活率〈44％，其差异有统计学意义（P〈0．05）。WesternBlot法检测结果显示，OCLN蛋白表达随着BPA染毒剂量的增加而降低，呈剂量依赖性，不同染毒剂量组与对照组比较，差异均有统计学意义（P〈0．05）。结论BPA可抑制睾丸支持细胞增殖活性和OCLN的表达，干扰支持细胞正常的生精过程。

Objective To investigate the mechanism of spermatogenesis damage by exploring the effect of bisphenol A （BPA） on the proliferative activity and expression ofoccludin （OCLN） of rat Sertoli cells. Methods Sertoli cells were isolated from the testicle tissues of male Wistar rats （20-22 days） and cultured in vitro, and then they were identified by rathonum red staining. Sertoli cells were divided into two groups： the BPA group （treated with 25 μM, 50 μM, 100 μM BPA for 24h）, and the solvent control group （no BPA）. The proliferative activity of Sertoli cells was measured by CCK-8 assay, and the expresssion of OCLN in Sertoli cells was detected by Western blot. Results Sertoli cells were isolated and cultured in vitro successfully, and the purification rate of which reached above 90%. The CCK- 8 assay showed that BPA inhibited the proliferation activity of Sertoli cells significantly. The survival rate decreased markedly, below 44% when the BPA concentration was larger than 103 μM. There was significantly statistical difference （P〈0.05）. The expression of OCLN was gradually decreased in parallel with increasing concentrations of BPA. Compared with that of the solvent control group, all the differences were significant （P〈0.05）. Conclusion BPAmight inhibit the proliferative activity and expression of OCLN of rat Sertoli cells, which suggests that BPA might interfere with the Sertoli cell＇ s function in spermatogenesis.