人羊膜间充质干细胞体外大量扩增的方法

摘要

BACKGROUND: Amnion mesenchymal-derived stem cells are obtained from the placenta with a placenta amniotic membrane of about 600 cm2.n OBJECTIVE: To establish a kind of simple isolation and culture method of mesenchymal stem cells derived from human amnion in vitro, and to explore their biological properties.n METHODS: Mesenchymal stem cells derived from human amnion were harvested by trypsin digestion combined with direct adherence. The morphology of human amnion-derived mesenchymal stem cells was observed. The passage 5 cells were collected to draw a cell growth curve. Surface markers and cell cycle of the passage 5 cells were determined using flow cytometry. Passage 4 cells were obtained for osteogenic and adipogenic induction. After 4 months of cryopreservation, the resuscitated passage 4 cells were counted to determine cell survival rate and draw the cell growth curve.n RESULTS AND CONCLUSION: A few of cells creped at day after primary seeding. About after 15 days, 80%-90% cells fused in a spindle shape. After passage, the cells showed even morphology and arranged spirally. The latency period of the passaged cells was 48 hours and logarithmic growth phase was about 4 days. After logarithmic growth phase, the cells entered the platform period. The flow cytometry results showed negative expression of CD34, CD14, HLA-DR, CD19, CD45, but positive expression of CD73, CD105, CD90 on the surface of mesenchymal stem cells. The alizarin red and oil red O staining was positive and confirmed osteogenic and adipogenic capacity of human amnion-derived mesenchymal stem cells. Flow cytometry results showed that 28% cells were in S phase. After cryopreservation, the survival rate of resuscitated cells was up to 90%, and the resuscitated cells had the same growth characteristic with the non-cryopreserved cells. These results confirm a simple method to proliferate a great amount of human amnion-derived mesenchymal stem cells.%背景：一个胎盘羊膜的面积约600 cm2，羊膜来源间充质干细胞即取材于胎盘上的羊膜。　　目的：建立一种简单的体外分离培养人羊膜来源间充质干细胞的方法，并分析其生物学特性。　　方法：采用胰酶和直接贴壁相结合的方法分离获取人羊膜来源间充质干细胞并进行体外培养，观察细胞形态，分析传代第5代细胞生长曲线，检测第5代细胞表面标志表达和检测细胞周期。取传代第4代细胞行体外成骨细胞诱导和成脂诱导，冻存第4代细胞6个月后复苏，计数复苏后细胞存活率并绘制复苏细胞生长曲线。　　结果与结论：原代接种后第9天有少许细胞爬出，15 d左右细胞达80%-90%融合，细胞以梭形为主。细胞传代后，细胞形态均一，螺旋状排列。传代细胞潜伏期48 h，对数增殖期4 d左右，对数增殖期后进入平台期。间充质干细胞表面CD34、CD14、CD19、CD45、HLA-DR 呈阴性表达，CD73、CD105、CD90呈阳性表达。茜素红染色及油红O染色阳性，证实具有向脂肪细胞、成骨细胞分化的能力。流式细胞术细胞周期检测，S期占28%。冻存复苏后细胞存活率达90%以上，且与未冻存传代细胞具有相同的生长特性。结果证实实验成功地建立了一种简化的人羊膜来源间充质干细胞大量扩增的方法。