Isolation,culture and identification of human amniotic-derived mesenchymal stem cells
Isolation,culture and identification of human amniotic-derived mesenchymal stem cells
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发表时间：
2006
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通讯作者：
Li Guoxi
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作者：
Li Guoxi
目的：探讨人骨髓间充质干细胞（AD-MSCs）的分离、培养及鉴定。方法：取足月剖宫产产妇羊膜。将收获的组织块机械切碎，并在37 ℃下用胰蛋白酶处理两次，持续30分钟。1 g/L），并在37°C下孵育60 min。用由DMEM/F12和10%胎牛血清组成的完全培养基培养和纯化细胞。相差显微镜下观察细胞形态学变化，流式细胞仪分析4~6代AD-MSCs的细胞表面抗原特性。RTRA和bFGF诱导AD-MSCs分化。结果：AD-MSCs在体外可增殖。流式细胞仪检测显示，CD 29、CD 44和HLA-A、B、C（MHC I类）表达阳性，CD 34、CD 45和HLA-DR（MHC Ⅱ类）表达阴性。经RTRA和bFGF诱导后，免疫细胞化学检测AD-MSCs表达神经元特异性标记物NSE。结论：羊膜中含有干细胞，在体外可快速扩增并分化为神经元样细胞。
Aim: To investigate the isolation, culture and identification of human amniotic-derived mesenchymal stem cells(AD-MSCs). Methods:The amniotic membrane was collected from full term cesarean section. The harvested pieces of tissue were mechanically minced and treated with trypsin twice at 37°C for 30 min. Then the tissue pieces were placed in DMEM/F12 containing collagenase (1.0 g/L) and DNase (0. 1 g/L) and were incubated at 37°C for 60 min. The cells were cultured and purified with complete medium consisting of DMEM/F12 and 10% fetal bovine serum. The morphological changes of the cells were observed under phase contrast microscope, and the cell surface antigenic characteristics of the cultured AD-MSCs at passage 4~6 were analyzed by flow cytometry. The differentiation of AD-MSCs were induced by RTRA and bFGF. Results: The AD-MSCs could proliferate in vitro. The flow cytometry analyses revealed that the expression of surface antigens, such as CD29, CD44 and HLA-A,B,C(MHC class I) was positive; but CD34, CD45, and HLA-DR(MHC class Ⅱ)were negative. After induced by RTRA and bFGF,AD-MSCs expressed the specific markers of neurons (NSE) by immunocytochemistry. Conclusion: The amniotic membrane contains stem cells that are capable of rapid expansion and differentiation into neuron-like-cells in vitro.