TGF-β1在大鼠睾丸支持细胞的作用及对紧密连接的影响  

Role of TGF-β1 in Sertoli cells and tight junction

作　　者：娄江涛[1] 魏任雄[1] 余亮亮[1] 陈建伟[1] 崔云[1] 

机构地区：[1]浙江中医药大学附属宁波市中医院男科实验室,浙江宁波315012

出　　处：《中华男科学杂志》2017年第6期510-516,共7页National Journal of Andrology

基　　金：浙江省宁波市自然科学基金项目(2016A610200)~~

摘　　要：目的:探讨TGF-β1在大鼠睾丸支持细胞增殖和凋亡中的作用以及对紧密连接相关蛋白基因表达的影响。方法:体外分离和培养大鼠睾丸支持细胞,分空白对照组、TGF-β1受体阻滞剂组、TGF-β1刺激组和TGF-β1+受体阻滞剂组共4组。CCK-8检测细胞增殖;流式细胞术检测细胞凋亡;建立支持细胞原代双室培养模型,应用跨上皮电阻测定法(TER)检测单层细胞两侧跨细胞电阻值;RT-PCR和Western印迹检测支持细胞闭合蛋白(Occludin)、闭锁小带蛋白1(ZO-1)和紧密连接蛋白Ⅱ(ClaudinⅡ)的相对表达量。结果:各组细胞增殖组间差异有统计学意义(F=5.05,P<0.05),TGF-β1刺激组与空白对照组相比细胞增殖率增加(P<0.05),而TGF-β1+受体阻滞剂组与TGF-β1刺激组相比细胞增殖率降低(P<0.05)。各组细胞凋亡率组间差异无统计学意义(F=1.13,P>0.05);各组细胞间TER差异有统计学意义(F=38.99,P<0.01),与空白对照组比较TGF-β1刺激组TER降低(P<0.01),与TGF-β1刺激组比较,TGF-β1+受体阻滞剂组和TGF-β1受体阻滞剂组细胞TER升高(P均<0.01);各组支持细胞中ZO-1、ClaudinⅡmRNA及蛋白在各组中的表达量差异均无统计学意义(F_(mRNA)=0.49,0.93,P均>0.05;F_(蛋白)=0.28,1.31,P均>0.05),而Occludin mRNA及蛋白在各组中的表达量差异均有统计学意义(F_(mRNA)=6.86,F_(蛋白)=6.87,P均<0.05);与空白对照组相比,TGF-β1刺激组Occludin mRNA及蛋白表达明显降低(P均<0.01),而加入受体阻滞剂后,Occludin mRNA及蛋白表达明显回升(P均<0.05)。结论:TGF-β1对睾丸支持细胞的增殖有促进作用,并能通过调节Occludin蛋白的表达而影响大鼠支持细胞间的紧密连接。Objective ： To explore the role of TGF-β1 in the proliferation and apoptosis of Sertoli cells and its effect on the ex- pressions of tight junction-related proteins and genes in rats. Methods： Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A （blank control） , B （TGF-β1 receptor blocker） , C （TGF-β1） , and D （TGF-β1 ＋ receptor blocker）. The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-cham- ber model for the primary culture of Sertoli cells, the transepithelia electrical resistance （TER） value was measured and the relative expressions of Occludin, ZO-1 and Claudin H determined by RT-PCR and Western blot. Results ： The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D （0.79± 0.04 vs 0. 66 ± 0.05 and 0.68 ± 0.02, P 〈 0.05 ） , with statistically significant differences among the four groups （ F = 5.05, P 〈 0.05 ）. However, no remarkable differencewith found among the four groups in the apoptosis rate of the cells （ F = 1.13, P 〉 0.05 ）. The TER value was dramatically decreased in group C as compared with groups A and D （ [ 176.37 ± 16.61 ] vs [ 281.42 ± 9.83 ] and [ 254.37 ± 13.55 ] cm2, P 〈 0. O1 ）, with statistieally signifieant differenees among the four groups （ F = 38.99, P 〈 0.01 ）. There were no remarkable differences among the four groups in the mRNA expressions of ZO-1 and Claudin Ⅱ （ F = 0.49 and 0.93, P 〉 0.05 ） or their protein expressions （ F = 0.28 and 1.31, P 〉 0.05 ）. Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D （ P 〈 0.01 and P 〈 0.05 ）, with statistically significant differences among the four groups （ F = 6.86 and 6.87, P 〈 0.01 ）. Conclu- sion ： TGF-β1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expres

关 键 词：转化生长因子Β1 睾丸支持细胞 紧密连接 闭合蛋白 大鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]