双酚A干扰大鼠生精过程时TJ渗透性屏障的体外研究(英文)  

BPA Disturb TJ- Permiablity of Rat Sertoli Cells During Spermatogenesis in Vitro

作　　者：冯友亮[1] 尹营营[1] 王新生[1] 王沛涛[1] 

机构地区：[1]青岛大学医学院附属医院泌尿外科,山东青岛266003

出　　处：《现代生物医学进展》2012年第8期1430-1434,共5页Progress in Modern Biomedicine

摘　　要：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。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.

关 键 词：双酚A 精子形成 紧密连接 支持细胞连接蛋白 

分 类 号：R698.21[医药卫生—泌尿科学]