CAJ | 학술논문

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。
배경：일개태반양막적면적약600 cm2，양막래원간충질간세포즉취재우태반상적양막。 　　목적：건립일충간단적체외분리배양인양막래원간충질간세포적방법，병분석기생물학특성。 　　방법：채용이매화직접첩벽상결합적방법분리획취인양막래원간충질간세포병진행체외배양，관찰세포형태，분석전대제5대세포생장곡선，검측제5대세포표면표지표체화검측세포주기。취전대제4대세포행체외성골세포유도화성지유도，동존제4대세포6개월후복소，계수복소후세포존활솔병회제복소세포생장곡선。 　　결과여결론：원대접충후제9천유소허세포파출，15 d좌우세포체80%-90%융합，세포이사형위주。세포전대후，세포형태균일，라선상배렬。전대세포잠복기48 h，대수증식기4 d좌우，대수증식기후진입평태기。간충질간세포표면CD34、CD14、CD19、CD45、HLA-DR 정음성표체，CD73、CD105、CD90정양성표체。천소홍염색급유홍O염색양성，증실구유향지방세포、성골세포분화적능력。류식세포술세포주기검측，S기점28%。동존복소후세포존활솔체90%이상，차여미동존전대세포구유상동적생장특성。결과증실실험성공지건립료일충간화적인양막래원간충질간세포대량확증적방법。
BACKGROUND: Amnion mesenchymal-derived stem cells are obtained from the placenta with a placenta amniotic membrane of about 600 cm2. 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. 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. 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.