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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。
AIM: To investigate the mechanism of SCJP injury induced by estrogen-BPA interference in vitro. Methods: Sertoli cells of Wistar rats were primarily cultured in vitro for 4-5 days. TJ permeable barrier was established by double chamber culture and the trans-epithelial resistance (TER) response was measured. The formation of the connection structure and the extent of BPA damage to the tight connection. Solvent (DMSO) as a negative control, to the final concentration of 25μM, 100μM of BPA on the supporting cells 24h, MTT assay of different concentrations of BPA role of Sertoli cells proliferation activity. Western bloting was used to observe the changes of occludin, ZO-1 and Cx43 expression. Results: Sertoli cells of Wistar rats were successfully isolated and cultured, and a good in vitro TJ barrier model was established. Dual cell culture support cell epithelial TER value reached its peak at d4, and then maintained a relatively stable state at d4-9. D4 was exposed to 200μM, 100μM and 25μM BPA, respectively. After 24, 48, 72, 96 (P <0.05). Compared with the DMSO solvent control group, the TER values of 200μM and 100μM BPA groups were significantly decreased (P <0.05) compared with the DMSO control group, while the TER values of 25μM BPA group had no significant changes (P> 0.05). MTT results showed that after treated with different concentrations of BPA for 24h, the absorbance (OD) of Sertoli cells gradually decreased with the increase of exposure dose. 102,103μM concentration group and solvent control group were significantly different (P <0.05), while the 10-2,10-1,100,101μM group and the solvent control group no significant difference (P> 0.05). The results of Western blot showed that the expressions of occludin, ZO-1 and Cx43 in each dose group were significantly lower than those in the solvent control group, respectively. The expressions of occludin and ZO-1 were decreased with the increase of the dose of 25, 50, 100, There was significant difference between the control group and the control group (P <0.05). There was also significant difference between the 100μM group and the 25μM group (P <0.05). Compared with the control group, the expression of Cx43 increased with the dose of 25μM (P> 0.05), but significantly increased in 100μM group (P <0.05). Compared with 25μM group, 100μM group was significantly increased (P <0.05). CONCLUSION: Bisphenol A can impair the normal permeability of TJ barrier by impairing the normal expression of connexin and thus affecting normal spermatogenesis.