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目的：研究体外大鼠睾丸支持细胞紧密连接蛋白（SCJP）在类雌激素- 双酚A(BPA) 干扰下的损伤机制。方法：对Wistar 大鼠睾丸支持细胞（Sertoli 细胞）离体原代培养4-5d，通过双室培养模型建立体外紧密连接（TJ）渗透性屏障，并测量其跨上皮电阻值（TER）反应紧密连接结构的形成及BPA 对紧密连接的损害程度。设溶剂（DMSO）做阴性对照, 以终浓度为25μM、100μM 的BPA 作用于支持细胞24h，MTT 法测不同浓度BPA 作用的Sertoli 细胞增殖活性。Western bloting 观察occludin、ZO-1、Cx43 表达的变化。结果：成功分离并培养Wistar 大鼠睾丸支持细胞，并建立良好的体外TJ 屏障模型。双室培养支持细胞上皮TER 值在培养的 d4 达到顶峰，然后在d4-9 维持相对较稳定的状态，d4 以200μM,100μM,25μM BPA 染毒，分别于染毒后24，48，72，96 和120h 测TER：与DMSO 溶剂对照组相比，200μM,100μM 的BPA 组TER 值明显下降（P＜0.05），而25μM 的BPA 组在染毒后TER 值无明显变化（P＞0.05）。MTT 结果显示：经不同浓度BPA 作用24h 后，Sertoli 细胞的吸光度（OD 值）随着染毒剂量的增加而逐渐降低。102 、103μM 浓度组与溶剂对照组有显著性差异（P＜0.05），而10-2、10-1、100、101μM 组和溶剂对照组无显著性差异（P＞ 0.05）。Western blot 结果显示：occludin、ZO-1、Cx43 在各剂量组均有表达，与溶剂对照组相比，occludin、ZO-1 表达均分别随作用剂量的增加而降低：25μM 组、100μM 组与溶剂对照组相比，差异均存在显著性（P＜0.05）；100μM 组与25μM 组相比，差异亦存在显著性（P＜0.05）。Cx43 的表达却随染毒剂量的增加而增加，与溶剂对照组相比，25μM 组表达无明显增加（P>0.05），而100μM 组则明显增加（P＜0.05）；与25μM 组相比，100μM 组表达明显增加（P＜0.05）。结论：双酚A 可通过损伤支持细胞连接蛋白正常表达，破坏了TJ 屏障渗透性，从而影响正常的精子形成过程。

英文摘要:

Objective: To investigate the effects of BPA exposure on the testicular expression of Sertoli cell junctional proteins (SCJP) in male rats during spermatogenesis. Methods: Primary sertoli cells were isolated and cultured from Wistar rat for 4-5 days, and the tight junction-permeability barrier was established by dual-chamber culture model. The effect of BPA on tight junctions was measured by the method of transepithelial electrical resistance (TER) assay. With a series of concentration BPA (0,25,and 100μM) co-incubating the Sertoli cells in vitro for 24h, cell proliferative activity was assessed with MTT assay, and the expression of occludin, ZO-1, Cx43 were determined by Western blotting. Results: BPA perturbs the integrity of the TJ-permeablity in Sertoli cells in vitro, which was associated with the decline of selected proteins at the tight junction, and gap junction at the blood-testis barrier. The expression of Cx43 increased while the expression of occludin and ZO-1 reduced in the Sertoli cell following BPA treatment. Conclusion: The present study showed that occludin, ZO-1 and specifically Cx43 could be early targets for BPA, which may be one of the contributing factors leading to impairment in spermatogenesis.

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