人脐带间充质干细胞与sertoli细胞共培养下向男性生殖细胞分化的研究  

Mediated differentiaiton of HUMSCs into male germ-like cells by co-culture with mouse sertoli cells

作　　者：谢丽春[1] 杨汉华[1] 赖秀蓝[2] 陈晓东[2] 陈业增 刘思征[2] 马廉[1] 

机构地区：[1]汕头大学医学院第二附属医院儿科,汕头515041 [2]深圳大学妇幼保健院,深圳市坪山新区妇幼保健院儿科,深圳518122

出　　处：《中国小儿血液与肿瘤杂志》2016年第6期293-299,共7页Journal of China Pediatric Blood and Cancer

基　　金：国家自然基金面上项目项目号:81070478;深圳市科创委项目(编号:CYJ20150402092905162);深圳市卫生计生系统科研(编号:201501053);广东省医学科学技术研究基金项目(编号:201512117919916)

摘　　要：目的探讨用睾丸支持细胞(SCs)模拟的睾丸微环境,通过共培养技术诱导人脐带间充质干细胞(HUMSCs)向男性生殖细胞分化的可能性,为男性不育治疗寻求一种新的途径。方法采用差异贴壁法分离、培养、扩增出HUMSCs,从新生昆明小鼠的睾丸中分离培养出SCs,制备SCs饲养层,将HUMSC:s加入SCs饲养层中共培养,显微镜观察诱导前及诱导后第7 d、14 d细胞的形态学变化;RT-PCR、细胞免疫荧光检测男性生殖细胞相关标记DAZL、VASA及Stella的表达。结果 HUMSCs在共培养体系中逐渐失去细小成纤维样的形状,形成类似精原细胞样的圆形细胞菌落,诱导7 d、14 d后,RT-PCR、细胞免疫荧光均可以检测到男性生殖细胞特异性标记VASA、DAZL及Stella的表达。结论在睾丸SCs模拟的睾丸微环境,HUMSCs不仅发生形态学的变化,而且能表达生殖细胞的特异生物学特性,表明在特定的微环境中,细胞与细胞间的相互接触是微环境中控制HUMSCs向男性生殖细胞方向分化的必不可少的因素。SCs与HUMSCs的直接接触诱导其向男性生殖细胞分化的诱导研究,为迫切需要的男性不育治疗提供了一个新的实验参考系统。Objective To investigate the effects of sertoli cells （SCs） in inducing HUMSCs differentiation into male germ cells by mimicked in vivo-like testicular niches. Methods HUMSCs was isolated and expanded by tissue adherent culture, primary SCs were prepared from decapsulated testes of newborn mice by sequential enzymatic digestion, and the HUMSC suspension was seeded on mitomycintreated SC monolayers to induce differentiation into germ cells. RT-PCR and immunofluorescence analysis of germ-like cells revealed expression of early germ-cell markers Stella and VASA and the male germ-cellspecific marker deleted in azoospermia-like （DAZL）. Results In the cocuhure system, HUMSCs lost their spindle-like shape, formed cell clusters, and assumed germ-cell shapes. HUMSCs expressing VASA, Stella and DAZL by reverse transcriptase-polymerase chain reaction and immunostaining. Conclusions Under appropriate condition, direct cell-to-cell contact between HUMSCs and sertoli cells is obligatory in relaying the external cues of the microenvironment controlling the differentiation of HUMSCs to male germ cells. These data indicated that HUMSCs differentiation depends on specific microenvironment, physical contact between HUMSCs and SCs and HUMSCs might be a promising source of cell transplantation therapy and a new way for infertility therapy. We might be able to identify specific substances regulating spermatogenesis under this coculture system in the future.

关 键 词：人脐带间充质干细胞 细胞间的直接接触 环境依赖性 男性生殖细胞 睾丸支持细胞 

分 类 号：R698.2[医药卫生—泌尿科学]