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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。
AIM: To investigate the effect of TGF-β1 on the expression of Cx43 in Leydig cells and the Cx43-mediated gap junctional intercellular communication (GJIC) in adult rats, and to investigate the effect of TGF-β1 on Leydig cells Whether it is related to changing the GJIC function between cells. Methods: Primary cultured and purified Leydig cells were divided into 6 groups: the experimental group were treated with 1, 2, 5 and 10 ng / ml of TGF-β1 for 20 h, and the blank control group were cultured in DMEM with 10% fetal bovine serum / F12 culture medium, and in some experiments, GJIC inhibitor Carbenox-olone was used as a positive control group. The localization and expression of Cx43 in cells were observed by immunofluorescence and Western blotting. Fluorescence bleaching recovery assay (FRAP) was used to detect the changes of GJIC function in cells. Results: The expression of Cx43 was speckled scattered in the cytoplasm and membrane of Leydig cells. After treated with TGF-β1 for 20 h, the expression level of Cx43 in the cytoplasm increased significantly with the increase of TGF-β1 concentration compared with the blank control group , While its expression in the cell membrane did not change significantly. Western blot results showed that the phosphorylation of Cx43 with the TGF-β1 concentration increased compared with the blank control showed a significant increase (P <0.05), while the non-phosphorylated state there was no significant difference. The results of FRAP showed that the intracellular fluorescence intensity was significantly decreased after treated with 5 ng / ml TGF-β1 for 20 h (P <0.01), and the average fluorescence recovery rate was only (43.58 ± 1.87)%. CONCLUSION: TGF-β1 can significantly down-regulate the GJIC function of Leydig cells. This inhibition may be achieved through increasing the expression of Cx43 in cytoplasm and increasing its phosphorylation level.