脐带间充质干细胞在精原细胞培养条件下的生殖细胞特性  

Biological Characteristics of Mesenchymal Stem Cells Cultured in Conditions for Spermatogonia in Vitro

作　　者：唐秋灵[1] 何红燕[2] 林广裕[1] 林丽敏[1] 谢庆东[3] 黄天华[3] 马廉[1] 

机构地区：[1]汕头大学医学院第二附属医院儿科,广东汕头515041 [2]汕头大学医学院第一附属医院外科,广东汕头515041 [3]汕头大学医学院生殖医学研究中心,广东汕头515041

出　　处：《实用儿科临床杂志》2008年第5期333-336,共4页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金项目资助(30772354);广东省社会发展计划项目资助(2007B080701022)

摘　　要：目的研究人脐带华尔通胶来源的间充质干细胞(MSCs)在精原细胞培养条件下向精原细胞方向分化的潜能。方法采用组织块贴壁法获得MSCs,取第3代MSCs分组进行诱导培养:对照组用基本培养液培养,实验组用条件培养液培养。用倒置显微镜和扫描电镜观察对照组和实验组细胞的外部形态差异;透射电镜观察2组细胞内部的超微结构变化;免疫组织化学方法检测2组细胞是否表达精原细胞的标记物CD117及CD49f;Western blot进一步检测2组细胞表达精原细胞特异性标记物CD49f的情况。结果以人脐带华尔通胶为原料培养获得的贴壁细胞高表达MSCs相关的标记,不表达或低表达造血细胞和与移植排斥相关的细胞表面标记,并可以维持在未分化状态稳定生长、增殖;实验组细胞形态发生了明显变化,由成纤维细胞形变为圆形,并发现极少数变圆的细胞继续分化呈形似蝌蚪状,对照组细胞形态不发生类似变化,超微结构也显示了很大的差异;免疫组织化学证实实验组细胞表达精原细胞的标记物CD117、CD49f,而对照组细胞不表达CD49f,CD117弱阳性表达;Western blot进一步证实人脐带MSCs诱导前不表达精原细胞标记CD49f,而诱导后表达CD49f。结论用组织块贴壁法获得人脐带来源的MSCs,在精原细胞培养条件下进行诱导培养,不仅能发生精原细胞样的形态变化及伴发细胞的分化过程,而且能表达精原细胞的特征性标记,证实人脐带来源的MSCs具有向精原细胞方向分化的潜能。Objective To investigate the possibility of inducing mesenchymal stem cells （MSCs） from human umbilical cord Wharton＇s Jelly to differentiate into spermatogonia. Methods To isolate, culture and purify MSCs with adherent method, the growth and proliferation of human umbilical cord - derived MSCs were observed, and their immunophenotypes were determined by flow cytometry ; MSCs of the third generation were divided into 2 groups to be induced and cultured , MSCs of the control group were cultured in basal medium, while those of the experimental group with conditional medium. The morphologic and ultrastructure changes of control group and experimental group cells were compared with phase contrast microscopy, electron microscopy （ EM ） and transmission electron microscope （TEM） respectively ; the spermatogonial cells differentiated were then evaluated by immunohistochemistry stained for CD117and CD49f ;the method of Western -blot was used to test if the cells induced could express CD49f. Results A population of MSCs were isolated from human umbilical Wharton＇s Jelly ; they were processed to obtain a fibroblast -like population of cells and could be maintained in vitro for extended periods with stable population doubling： After induction , the shape of MSCs changed greatly from the fibroblast to the round, even familiar to the tadpole ; expressed the known molecular markers of spermatogonial cells, such as CD49f, CD117. Conclusion The induced MSCs not only undergo spfermatogonial -cell like morphologic changes, ultramicrostructure mature with increasing cell organs, but also express the spermatogonial cell markers, which show that human umbilical cord derived MSCs are capable of differentiating into spermatogonial cell.

关 键 词：脐带 间充质干细胞 精原细胞 生物学特征 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]