用于人类脂肪干细胞的纯化和体外长期培养的聚合物涂层

Polymer Coatings for Purification and Long-term Culture of Human Adipose-derived Stem Cells

采用紫外固化法制备了基于丙烯酸酯类水凝胶的聚合物涂层(PC),并用X射线光电子能谱(XPS)、水接触角(WCA)和原子力显微镜(AFM)分别对PC进行了化学组成和表面性能的表征.在PC表面进行了人类脂肪干细胞(h ASC)的体外长期培养扩增,得到的第3代细胞的生物学表征结果表明,干细胞在PC表面能正常黏附生长,流式细胞仪检测发现干细胞对特征标记物CD49d,CD73,CD105的阳性显性比例较高,对HLA-DR和CD31几乎不显性,说明扩增的干细胞具有h ASC特征.对PC上扩增的干细胞进行诱导分化,并用油红O、茜素红和阿利新蓝分别进行染色分析,结果表明,该干细胞保留了h ASC的多能特性:能分化为成脂、成骨和成软骨细胞.含有单体甲基丙烯酰氧乙基三甲基氯化铵(DMC)、甲基丙烯酸环己酯(CHMA)和甲基丙烯酸-2-(二乙氨基)乙酯(DEAEMA)的PC2(质量比为3∶1∶2)在用于h ASC体外长期培养时,比其它PC和TCP更有利于细胞的黏附和增殖,纯化细胞,保持其多能性.实时荧光定量PCR(RT-q PCR)的分析表明PC2上得到的细胞更容易向成骨和成软骨细胞分化.

Polymer coating（ PC） based on polyacrylate hydrogels was prepared through UV initiated polymerization and characterized with X-ray photoelectron spectroscopy（ XPS）,water contact angles（ WCA）and atomic force microscopy（ AFM） respectively to understand their chemistry and surface properties. The biological characteristics of human adipose-derived stem cell（ h ASC） were evaluated after long-term cultured on PC in vitro for 3 passages. The results indicated that the h ASC proliferated successfully on the PC,expressed high percentage of specific membrane markers CD49 d,CD73,CD105,and very low percentage of HLA-DR and CD31 by using flow cytometry. The results suggested that the proliferated stem cells had the characteristics of h ASC. The h ASC were differentiated to adipocytes,osteoblasts and chondrocytes and stained with Oil Red O,Alizarin Red S and Alcian Blue respectively to demonstrate the maintaining of pluripotency of h ASC. The results showed that the PC2 based on methacrylatoethyltrimethyl ammonium chloride,cyclohexyl methacrylate and diethyl amino ethyl methacrylate（ mass ratio of 3 ∶ 1 ∶ 2） was the best in h ASC binding and proliferation,improved the purification,maintained the pluripotency among the three coatings and TCP. Relative gene expression of the differentiated cells was analyzed by quantitative real-time polymerase chain reaction（ RT-q PCR）. The results show that the harvest h ASC from PC2 are apt to differentiate to osteoblasts and chondrocytes.