诱导人孤雌胚胎干细胞向类间充质干细胞分化的实验研究  

Experimental Study of Inducing Human Parthenogenetic Embryonic Stem Cells to Differentiate into Mesenchymal Stem-like Cells

作　　者：艾爱[1] 陈瑶[1] 唐郑雅[1] 宋楠[1] 曹谊林 张文杰 

机构地区：[1]上海交通大学医学院附属第九人民医院辅助生殖科,上海市200011 [2]上海组织工程研究重点实验室,上海市200011

出　　处：《组织工程与重建外科杂志》2010年第3期128-131,共4页Journal of Tissue Engineering and Reconstructive Surgery

基　　金：国家重点基础研究发展计划(2005CB522705);国家自然科学基金(30571925;30671051)

摘　　要：目的探索人孤雌胚胎干细胞在体外向类间充质干细胞诱导分化的方法 ,并鉴定所得细胞的生物学特性。方法人孤雌胚胎干细胞在无血清条件下悬浮培养,形成拟胚体,10d后在含血清条件下使拟胚体贴壁生长,7d后胰酶消化,所得细胞在含血清的培养液中传代、扩增。观察传代、扩增后细胞的形态学变化;用免疫荧光染色和流式细胞技术进行细胞表型分析;取第9代细胞进行成脂、成骨和成软骨诱导,9～28d后行特殊染色及RT-PCR分析。结果人孤雌胚胎干细胞在诱导分化后,形态与骨髓间充质干细胞相似,多次扩增传代后仍保持细胞形态和扩增能力。免疫荧光染色发现,细胞表达中胚层标志波形蛋白(Vimentin)。流式细胞分析显示,细胞表达CD29、CD105、CD166、CD44等间充质干细胞表面标志。特殊染色及RT-PCR分析显示:成骨诱导后,细胞碱性磷酸酶和茜素红染色阳性,碱性磷酸酶和Cbfa-1表达增加;成软骨诱导后,细胞Ⅱ型胶原染色阳性,Ⅱ型胶原和软骨寡聚基质蛋白(COMP)表达增强;成脂诱导后,细胞油红染色阴性,脂蛋白酶和Leptin无表达。结论人孤雌胚胎干细胞可以诱导、分化为间充质干细胞,并具有成骨、成软骨分化潜能。Objective To explore a method of induced differentiation from human parthenogenetic embryonic stem cells （hPESCs） to mesenchymal stem-like cells, and identify the biological properties of the cells. Methods The hPESCs were cultured via suspension method to form embryoid bodies （EBs） in serum-free media for 10 days. EBs were then attached to the culture dish in serum-containing media. After 7 days of adherent culture, the cells were digested by pepsin and replaced for further expansion. The morphological features were observed during the passages. After 9 passages, the phenotype was analyzed by immunofluorescent staining and flow cytometry. Cells of the 9th passage were induced by osteogenic, adipogenic, and chondrogenic media. The induced cells were analyzed by specific staining and RT-PCT 9-28 days later. Results After induction, the hPESC-derived cells expanded stably and exhibited similar morphology to bone marrow mesenchymal stem cells （BMSCs）. Immunofluorescent staining revealed that the cells expressed vimentin, a mesoderm marker. Flow cytometry revealed that the cells expressed the surfaces markers of BMSCs, including CD29, CD44, CD105 and CD166. After osteogenic induction, the cells showed positive results in alkaline phosphatase and alizarin red staining. Increased expression of alkaline phosphatase and Cbfa-1 were observed in RT-PCR. After chondrogenic induction, the cells showed positive result of type Ⅱ collagen staining, and increased expression of type Ⅱ collagen and cartilage oligomeric matrix protein （COMP） expression in RT-PCR. After adipogenic induction, however, the cells showed negative results in oil red staining, and no expression of lipoprotein lipase and leptin in RT-PCR. Conclusion After induction, hPESCs can differentiate into mesenchymal stem-like cells with osteogenic and chondrogenic potential.

关 键 词：人孤雌胚胎干细胞 间充质干细胞 分化 

分 类 号：Q813.11[生物学—生物工程]