人胎盘羊膜上皮细胞的分离鉴定及多向诱导分化

摘要

背景:人羊膜上皮细胞来源稳定,正逐渐成为广受关注的再生医学种子细胞来源.目的:建立人羊膜上皮细胞体外分离培养、成脂、成软骨、成骨诱导分化的方法.方法:通过胰蛋白酶消化法从人胎盘羊膜中分离羊膜上皮细胞,进行体外培养与鉴定,观察培养12 d内的细胞生长曲线.取P1代羊膜上皮细胞,分别进行成脂、成软骨、成骨诱导,以常规培养细胞为对照,诱导16 d后,分别进行油红O染色、Masson染色及碱性磷酸酶染色,同时采用荧光定量PCR检测诱导过程中成脂转录因子、Ⅱ型胶原蛋白、骨桥蛋白、碱性磷酸酶mRNA表达变化.结果与结论:①从人羊膜中分离出的羊膜上皮细胞,免疫荧光检测表达上皮细胞标记物CK19;②P1代细胞具有较强的分裂增殖能力,P2细胞与P1相比增殖能力略有下降,P3细胞增殖能力最差;③羊膜上皮细胞经成脂、成软骨、成骨诱导后,油红O染色有红色脂滴,Masson染色有亮蓝色软骨基质,碱性磷酸酶染色有红褐色钙结节;随着诱导时间的延长,成脂转录因子、Ⅱ型胶原蛋白、骨桥蛋白、碱性磷酸酶表达量升高;④结果表明,采用酶消化法可从人羊膜中分离得到羊膜上皮细胞,羊膜上皮细胞可向脂肪细胞、成软骨细胞、成骨细胞分化.%BACKGROUND: Human placenta is a stable source for human amniotic epithelial cells, which is becoming a cellsource in the regenerative medicine that attracts widespread attentions.OBJECTIVE: To establish the method of isolation, culture, and adipogenic, chondric and osteogenic differentiationof human amniotic epithelial cells. METHODS: Trypsin-EDTA digestion was used to isolate human amniotic epithelial cells from human amnion tissue,which were then cultured and identified in vitro. The growth curve of the cells was observed in 12 days. Passage 1human amniotic epithelial cells were induced to differentiate into adipocytes, chondrocytes and osteoblasts, andconventional cultured cells were used as controls. After 16 days induction, oil red O, Masson and alkaline phosphatesstaining methods were carried out, and adipogenic transcription factor, type Ⅱ collagen, osteopontin, alkalinephosphatase mRNA expressions were detected using real-time fluorescene quantitative PCR.RESULTS AND CONCLUSION: Human amniotic epithelial cells were successfully obtained from human amnion tissue.Immunofluorescence data showed the expression of epithelial cell surface marker CK19. Passage 1 cells had a strongability to divide and proliferate. Compared with passage 1 ones, passage 2 cells showed a slight decrease in proliferationability, and the proliferation ability of passage 3 cells was the worst. Red lipid droplets, brilliant blue cartilage matrix andreddish brown calcium nodes were detected by oil red O, Masson and alkaline phosphates staining after adipogenic,chondrogenic and osteogenic differentiation, respectively. With the time prolonged, the expressions of adipogenictranscription factor, type Ⅱ collagen, osteopontin and alkaline phosphatase mRNA were increased. These resultsdemonstrated that human amniotic epithelial cells could be isolated from human amniotic membrane by enzymedigestion method, and these amniotic epithelial cells could be induced to differentiate into differentiate into adipocytes,chondrocytes and osteoblasts.