成体组织来源的Sca-1^+CD117-Lin-多潜能干细胞生物学特性分析

Biological characteristics of Sca-1^+CD117-Lin-pluripotent stem cells derived from adult tissues

背景:关于成体干细胞可塑性机制的解释,目前多数学者接受的观点是多种组织器官内存在的成体干细胞其实并非均一的细胞群体,其中包含着更为原始的多潜能干细胞,但对于多潜能干细胞的起源、鉴别及其生物学特性仍缺乏了解。目的:探讨从成体组织中能否分离出一群具有独特表型的更为原始的多潜能干细胞。设计、时间及地点:细胞学体外观察,于2007-03/2008-01在北京协和医学院基础学院组织工程中心完成。材料:清洁级12.5~14.5dBALB/C孕鼠10只。方法:无菌条件下打开BALB/C孕鼠子宫获取胚胎体壁组织,胰蛋白酶消化后贴壁法制成细胞悬液,接种后免疫磁珠分选Sca-1+CD117-Lin-细胞群,当细胞达70%~80%融合时消化传代,取第5代细胞用于实验。主要观察指标:倒置相差显微镜和扫描电子显微镜观察该类细胞的显微和超微形态,通过生长曲线和细胞周期观察其基本生长特性,采用流式细胞技术分析该群细胞免疫表型,以RT-PCR法检测胚胎干细胞相关抗原的表达情况,通过VonKossa染色、油红O染色、甲苯胺蓝染色及免疫细胞荧光染色观察其向三胚层细胞分化的潜能。结果:Sca-1+CD117-Lin-细胞贴壁生长,呈纺锤或短梭形,可在体外快速稳定扩增50代以上;传代后3~7d为对数生长期,此后即进入平台期生长,倍增时间约为32h,90.43%细胞处于G0/G1期,G2/M+S期的细胞比例为9.57%;细胞表型稳定,高表达Sca-1,不表达CD117,CD14,CD19,CD31,CD34,CD45和Flk-1,中度表达CD44;胚胎干细胞相关抗原Sox2,Oct-4及Nanog均呈阳性表达;诱导分化实验表明该细胞可以向中胚层来源的成骨细胞、脂肪细胞、软骨细胞、外胚层来源的神经胶质细胞以及内胚层来源的肝细胞分化。结论:从成体组织内可以分离出Sca-1+CD117-Lin-细胞群,其表达胚胎干细胞相关抗原,能够向三胚层分化,是一群更为原始的多潜能成体干细胞。

BACKGROUND： The mostly accepted standpoints regarding adult stem cell plasticity are that adult stem cells existing in various organs contain primitive pluripotent stem ceils. However, the origin, identification methods and biological characteristics of pluripotent stem cells remain unclear. OBJECTIVE： To investigate if a kind of pluripotent stem cells with more primitive character can be isolated from adult tissues. DESIGN, TIME AND SETTING： The in vitro cytology observation was performed at the Center of Tissue Engineering, Basic Medical Department of Peking Union Medical College from March 2007 to January 2008. MATERIALS： Pregnant BALB/C rats on gestation day of 12.5-14.5 d were adopt in the study. METHODS： The fetal perisome samples were attained from pregnant BALB/C rats under sterile conditions, and made into cell suspension by trypsin digestion method. Immunomagnetic beads were used to sort Sca-1^＋CD117Lin- cells. When 70%-80% cells were mixed, the cells were subcultured, and the 5 passage cells were taken in the experiment. MAIN OUTCOME MEASURES： Microstructure, ultrastructure, growth characteristics, immunophenotype and embryonic stem cell associated antigen were analyzed by inverted phase contrast microscope, scanning electron microscope, flow cytometry and RT-PCR respectively. Histochemical and immunofluorescence staining were used to investigate the potentiality of the Sca-1^＋CD117^-Lin^-cells differentiated into trilaminar germ disc. RESULTS： Sca-1^＋CD117Lin^- cells adhering to the wall with spindle or short fusiform shapes, which could amplification fast and stablely to 50 passages. Cells were found logarithmic growth on days 3-7, after that cell growth reached platform stage. There were about 32 h for doubling time, 90.43% ceils were at the G0/G1 phase, and 9.57% at the G2/M＋S phase. The cells expressed high levels of Sca- 1, no levels of CD 117, CD 14, CD 19, CD31, CD34, CD45 and Flk- 1, and moderate levels of CD44. The phenotype of human BMSCs was stabilization. Embryonic stem cell associated antigen Sox2, Oct-4 or Nanog were showed positive expression. RT-PCR indicated that the cells were able to differentiate into osteoblasts, adipocytes, chondrocyte, hepatocyte-like cells and neuroglia cells in vitro. CONCLUSION： The Sca-1^＋CD117Lin^- ceils isolated from adult tissue are primitive multiple potential adult stem cells, which can differentiate into trilaminar germ disc.