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目的探讨Wnt/β-catenin信号通路在成人睾丸支持细胞增殖中的作用及其作用机制。方法取成人睾丸组织采用两步酶法消化,分离纯化支持细胞,后行传代培养,并经波形蛋白免疫荧光染色鉴定其纯度。将支持细胞分为3组:正常对照组(Con组)、添加Gsk-3β特异性抑制剂SB216763组(SB组)和添加氯化锂组(LiCl组)。通过BrdU标记和流式细胞仪检测细胞增殖和细胞周期;采用免疫荧光染色、实时荧光定量PCR和Western blot-ting检测细胞中β-catenin和c-myc的表达状况;采用RNA干扰技术,下调支持细胞中c-myc基因表达。结果BrdU标记和流式细胞仪检测结果显示,SB和LiCl组支持细胞增殖明显高于Con组;加入SB216763和LiCl可明显促进细胞β-catenin和c-myc入核,c-myc mRNA和蛋白水平明显高于Con组;转染c-myc siRNA后,可明显降低SB216763引起的支持细胞增殖。结论激活Wnt/β-catenin信号通路可促进成人睾丸支持细胞的增殖,其作用机制可能是通过上调c-myc基因表达来实现的。
Objective To investigate the role of Wnt / β-catenin signaling in the proliferation of adult human testis-supporting cells and its mechanism. Methods The adult testis was digested by two-step enzymatic digestion, and the supporting cells were isolated and purified. The cultured cells were subcultured and identified by vimentin immunofluorescence staining. The supporting cells were divided into 3 groups: control group (Con group), SB216763 (SB group) and LiCl group (Gsk-3β specific inhibitor). The cell proliferation and cell cycle were detected by BrdU labeling and flow cytometry. The expression of β-catenin and c-myc in cells were detected by immunofluorescence staining, real-time fluorescence quantitative PCR and Western blot-ting. C-myc gene expression in cells. Results The results of BrdU labeling and flow cytometry showed that the proliferation of SB and LiCl groups was significantly higher than that of Con group. The addition of SB216763 and LiCl significantly enhanced the expression of β-catenin and c-myc, c-myc mRNA and protein Which was significantly higher than that of Con group. The transfection of c-myc siRNA could significantly reduce the proliferation of SB216763-induced cells. Conclusion Activation of Wnt / β-catenin signaling pathway can promote the proliferation of adult human testicular sertoli cells. The mechanism may be through the up-regulation of c-myc gene expression.