Wnt/β-catenin信号通路在成人睾丸支持细胞增殖中的作用及其机制  

Effect and mechanism of the Wnt/β-catenin signaling pathway on proliferation of adult human Sertoli cells

作　　者：李毅[1] 张帅[2] 殷钢[3] 史琨[1] 邴鲁军[1] 郝晶[1] 

机构地区：[1]山东大学医学院组织胚胎学教研室,济南250012 [2]泰安市中心医院生殖中心,山东泰安271000 [3]山东大学齐鲁医院泌尿外科,济南250012

出　　处：《山东大学学报（医学版）》2012年第3期55-61,共7页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30871405;30971653);山东省自然科学基金资助课题(ZR2009CM018)

摘　　要：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 effect and mechanism of Wnt / β-catenin signaling pathway on adult human Sertoli cell proliferation.Methods Adult testicular tissues were digested by the two-step enzymatic method to extract and purify Sertoli cells,and the purified Sertoli cells were continuously passage cultured.The Sertoli cell-specific protein vimentin was detected by immunofluorescence to evaluate the purity of the cultured Sertoli cells.The cultured Sertoli cells were divided into three groups： the normal culture medium（Con group）,the medium with Gsk-3β inhibitor SB216763（SB group）,and the medium with lithium chloride（LiCl group）.BrdU incorporation assay was performed to measure Sertoli cell proliferation and flow cytometry was used to access the cell cycle.Immunofluorescence,real-time fluorescent quantitative PCR and Western blotting were performed to investigate expressions of β-catenin and c-myc.C-myc expression was down regulated in Sertoli cells by small RNA interference technology.Results BrdU incorporation assay and flow cytometry results revealed that SB216763 and the LiCl significantly promoted Sertoli cell proliferation.The nuclear translocation of β-catenin and c-myc,the mRNA level and protein expression of c-myc remarkably increased in SB group and the LiCl group.The proliferation stimulation effect of SB216763 on human Sertoli cells was significantly inhibited after c-myc siRNA transfection.Conclusion Activation of the Wnt/β-catenin pathway could promote the proliferation of the adult testicular Sertoli cells via up-regulation of c-myc expression.

关 键 词：成人 睾丸 支持细胞 增殖 Β-CATENIN C-MYC 

分 类 号：R737.21[医药卫生—肿瘤]