摘要
目的 探讨体外维甲酸联合异位重构生精小管诱导小鼠诱导多能干细胞（iPSCs）向雄性生殖细胞分化的潜能. 方法 自2010年12月至2011年12月,采用悬浮法培养小鼠iPSCs形成类胚体.类胚体培养至第5天,添加维甲酸进行诱导,不添加维甲酸的类胚体自发分化组作为对照组.收集培养10d的类胚体,采用实时定量PCR和免疫细胞化学检测其生精细胞相关基因和蛋白的表达.同时将类胚体消化为单细胞,与新生小鼠睾丸细胞混合注入雄性裸鼠皮下,移植完成后裸鼠行手术去势,移植后第4、6、8、12周取移植物进行HE染色和免疫组织化学染色. 结果 小鼠iPSCs经类胚体形成及维甲酸诱导后Stra8、Odf2、Act和Prm1基因的相对表达量上调,分别为对照组的（4.50±1.02）、（1.48±0.04）、（10.52±0.25）和（323.28±25.64）倍;Oct-4、Dppa3、Piwil2、Tex14和Scp3基因相对表达量下调,分别为对照组的0.56±0.02、0.11±0.01、0.50±0.01、0.53±0.14和0.16±0.00.类胚体同时表达雄性生殖细胞相关蛋白VASA和联合复合体蛋白3.HE染色发现,iPSCs来源生精细胞和新生小鼠的睾丸细胞共移植后能重构成类生精小管.免疫荧光染色结果显示,类生精小管管腔内可见由iPSCs分化而来的VASA阳性生精细胞. 结论 小鼠iPSCs经类胚体形成和维甲酸诱导后可分化为雄性生殖细胞.部分iPSCs来源的生精细胞可定居于类睾丸生精小管的基底膜,异位重构的生精小管可为iPSCs向雄性生殖细胞分化提供适宜的微环境.
Objective To investigate the differentiation potential of induced pluripotent stem cells （iPSCs） into male germ cells through retinoic acid （RA） induction in vitro and ectopic transplantation in vivo.Methods Mouse iPSCs were first transferred to bacterial plates to form embryoid body （EB）.After 5days,EB was treated by 2 μmol/L RA.After 10 days of cuhure,iPSCs-derived cells were dissociated from EB.The real-time PCR and immunocytochemical assay were used to detect the expression of related gene and protein.Then,they were injected into the male nude mice with testicular cells from neonatal mice.The grafts were obtained for further HE and immunohistochemical staining on 4,6,8,12 weeks after injection.Results After EB formation and RA induction,the expressions of Stra8,Odf2,Act,and Prm1 were upregulated in induction group,which were 4.50± 1.02,1.48±0.04,10.52±0.25,323.28±25.64 times as control group,respectively.On the contrary,the expressions of Oct-4,Dppa3,Piwil2,Tex14,and Scp3 were down-regulated in induction group,which were 0.56±0.02,0.11±0.01,0.50±0.01,0.53±0.14,0.16±0.00times as control group,respectively.Meanwhile,RA-treated EBs also expressed male germ cell markers VASA and SCP3.HE staining showed that the seminiferous tubules could be reconstituted after injecting the iPSCs-derived cells and testicular cells from neonatal mice.Immunofluorescence analysis revealed co-localization of EGFP-positive and VASA-positive cells in the reconstituted tubules.Conclusions Mouse iPSCs could differentiate into male germ cells with EB formation and RA induction.The iPSCs-derived germ cells could settle in the basement membrane of reconstituted tubules,which indicates reconstituted seminiferous tubules may provide a functional niche for exogenous iPSCs-derived male germ cells.
出处
《中华泌尿外科杂志》
CAS
CSCD
北大核心
2014年第4期306-311,共6页
Chinese Journal of Urology
基金
国家自然科学基金（31201109）
国家重点基础研究发展计划（973计划）（2010CB945200）
关键词
诱导多能干细胞
类胚体
维甲酸
异位重构
雄性生殖细胞
Induced pluripotent stem cells
Embryoid body
Retinoic acid
Ectopic reconstitution
Male germ cells
