人羊膜间充质干细胞的生物学特性鉴定

Characterization of surface markers of human anmion mesenchym-derived side population cells

目的分离提纯的人羊膜间充质干细胞的生物学特性鉴定。方法剥去38～40周孕妇剖腹产术后羊膜，用消化酶消化后制备人羊膜间充质细胞（hAMCs）悬浮液。利用流式细胞分选技术从hAMCs中分离提纯干细胞（hAMC-SP细胞），并对于细胞抗原进行鉴定。结果从hAMCs中分离的hAMC-SP细胞，约占AMCs总数的0．3％。传代培养4～10代后的hAMC-SP细胞表达Nestin、Vimentin、整合素家属成员（CD49b、CD49c、CD49d、CD49e）、CD9、CD13、CD19、CD29、CD44、CD46、CD51、CD59、CD166及干细胞相关的Oct-3／4抗原。HLA-ABC、TRA-1-81及SSEA-4为弱表达；相反CD34、CD45、CD117、CD56、CD90、CD105、CD106、CD133、Fit-1、Musashi 1及HLA-DR无阳性结果，同样TRA-1-60及SSEA-3也表达阴性。体外转化实验表示hAMC-SP细胞在琼脂糖培养基上不形成细胞集落，但阳性对照组的HepG2形成多数细胞集落。结论hAMC-SP细胞表面标记符合骨髓及脂肪间充质等来源的干细胞特点；hAMC-SP细胞迅速而稳定扩增，无致瘤性。由于来源充足，hAMC-SP细胞在消除HLA-ABC阳性细胞亚群下，同种异体移植在内的广泛的再生医学领域中能成为理想的细胞来源。

Objective We aim to characterize the side population cells obtained by FACS from human amnion cells （hAMC） as stem cells. Methods hAMC were prepared by an enzymatic treatment from the amniotic membrane separated mechanically from the placenta obtained by Caesarean section of 38-40 weeks after pregnancy. SP cells（hAMC-SP） were isolated by FACS from them and studied for stem cell antigens. Results hAMC-SP cells were about 0.3% among hAMCs. The SP cells at fourth to tenth passages after the culture start were positive for Nestin, Vimentin, integrin family antigens （CD49b,CD49c .CD49d ,CD49e） and other antigens such as CD9, CD13, CD19, CD29, CD44, CD46, CD51, CD59, CD166, and Oct-3/4. HLA-ABC,TRA-1-81 and SSEA-4 were weakly positive. In contrast, CD34, CD45, CD117, CD56, CD90, CD105, CD106, CD133, Fit-1, Musashi and HLA-DR were negative. Similarly, TRA-1-60 and SSEA-3 were negative. The in vitro transformation assay revealed that hAMC-SP cells did not form colonies in soft agarose cultures, whereas HepG2 cells, used as the positive controls of transformed cells, formed many colonies. Conclusion The cell surface marker studies suggest that hAMC-SP share characteristics with the stem cells obtained from other organs such as bone marrow mesenchymal cells and fat tissues, hAMC-SP cells rapidly and stably proliferate without transforming and they indeed differentiate into several cell types upon exposure to differentiation media. These results suggest that hAMC- SP, after proper depletion of HLA-ABC positive populations, can be used as ideal stem cells in a wide range of regenerative medicine over allogenicity because of their abundant presence.