TGF-β1对大鼠睾丸Leydig细胞间连接蛋白43介导缝隙连接通讯功能的影响

TGF-β1 reduces connexin43-mediated gap junctional intercellular communication in rat Leydig cells

目的:观察转化生长因子TGF-β1对成年大鼠睾丸Leydig细胞间连接蛋白Cx43表达以及由Cx43介导的缝隙连接细胞间通讯(GJIC)功能的影响,旨在探讨TGF-β1对Leydig细胞的影响是否与其改变细胞间GJIC功能相关。方法:将原代培养纯化的Leydig细胞分为6组:实验组分别以1、2、5、10 ng/ml的TGF-β1处理细胞20 h,空白对照组以含10%胎牛血清的DMEM/F12培养液处理细胞,部分实验中以GJIC抑制剂甘珀酸(Carbenox-olone)处理细胞作为阳性对照组。采用免疫荧光法和Western印迹观察Cx43在细胞中的定位和表达变化,用荧光漂白恢复实验(FRAP)检测细胞间GJIC功能的改变。结果:Cx43表达呈斑点状散在分布于Leydig细胞的胞质和胞膜中,TGF-β1处理20 h后,Cx43在胞质中的表达强度随着TGF-β1浓度的增加较空白对照组明显增强,而其在胞膜中的表达则无明显改变。Western印迹结果显示磷酸化状态的Cx43随着TGF-β1浓度增加表现出较空白对照明显增强的趋势(P<0.05),而非磷酸化状态则无显著差异。FRAP结果显示经5 ng/ml TGF-β1作用20 h后细胞内荧光强度较空白对照明显减弱,具有显著性差异(P<0.01),且其平均荧光恢复率仅为(43.58±1.87)%。结论:TGF-β1可显著下调Leydig细胞间的GJIC功能,这种抑制作用可能通过增加其连接蛋白Cx43在细胞质中的表达,提高其磷酸化水平来实现。

Objective： To observe the effects of TGF-β1 on the expression of connexin43（Cx43） and Cx43-mediated gap junctional intercellular communication（GJIC） in rat Leydig cells,and investigate the association of its effects on Leydig cells with its ability of changing GJIC.Methods： Primarily cultured purified Leydig cells were divided into a blank control group,a positive control group（treated with the GJIC inhibitor Carbenoxolone）,and four TGF-β1 groups（treated with TGF-β1 at the concentration of 1,2,5 and 10 ng/ml,respectively,for 20 hours）.The localization and expression of Cx43 were detected by immunofluorescence and Western blot,and the changes in GJIC analyzed by FRAP assay.Results： Cx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells.TGF-β1 significantly elevated the expression of Cx43 in the cytoplasm,but caused no evident change in the membrane.Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-β1 as compared with that of the blank control group（P0.05）.After 20 hours of treatment with TGF-β1 at 5 ng/ml,the fluorescence intensity of Leydig cells was markedly reduced（P0.01）,with a mean fluorescence recovery rate of merely（43.58±1.87）%.Conclusion： TGF-β1 could significantly down-regulate GJIC between adjacent Leydig cells,and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.