不同培养条件下脂肪干细胞及软骨细胞共培养探究.docVIP

不同培养条件下脂肪干细胞及软骨细胞共培养探究 　【摘要】 探讨病理状态下软骨细胞能否诱导脂肪干细胞(ADSC)向软骨细胞分化以及可能的最佳条件，以便为临床修复关节软骨损伤提供可能的新途径。［方法］分离共培养新西兰大白兔骨关节炎模型的ADSC和软骨细胞，根据不同血清浓度（10％ FBS和2％ FBS）和不同培养空间（纤维蛋白凝胶支架和无支架单层培养）分组，共培养14 d后，胰酶消化终止。倒置显微镜观察和透射电镜观察共培养后ADSC的形态变化。甲苯胺蓝染色法和免疫细胞化学检测共培养后ADSC的蛋白多糖和Ⅱ型胶原的表达水平。RTPCR检测蛋白多糖和Ⅱ型胶原的基因转录水平。［结果］共培养7 d后部分ADSC变圆，14d时ADSC形态高度分化与成熟软骨细胞相似，其蛋白多糖和Ⅱ型胶原的基因转录和蛋白表达均增高，尤以10％FBS支架共培养组最为明显。［结论］与病理状态下软骨细胞共培养后，ADSC可以被诱导成软骨样细胞。高浓度血清三维培养可以增强这种诱导作用。 【关键词】 脂肪干细胞； 软骨细胞； 共培养； 组织工程 Abstract：［Objective］To investigate the feasibility and optimal condition of in vitro chondrogenesis by coculture of adipose derived stem cell (ADSC) and chondrocytes in pathologic state so as to provide a new clinical approach for repairing damaged articular cartilage.［Method］ADSC and aricular chondrocytes from osteoarthritis model in adult New Zealand white rabbits were in vitro expanded and then seeded on plate and plugin type millicell dish respectively. The coculture cells were cultured in different conditions including different serum concentration (10% FBS and 2% FBS) and dimensions (fibrin scaffold and monolayer). The culture medium was changed every 3 days. The shape of ADSC before and after coculture was observed by inverted microscope and transmission electron microscope. The expression of aggrecan and type Ⅱ collagen genes in ADSC were studied by toluidine blue staining and immunohistochemistry after in vitro coculture for 14 days. And the transcription of aggrecan and type Ⅱ collagen genes in ADSC were studied by RTPCR. ［Result］The shape of ADSC was fibroblastlike cells morphologically. And they became round at 7 days after in vitro coculture. At 14 days after coculture with chondrocytes, ADSC was changed to chondrocytelike cells morphologically and increased immunostaining particles of type Ⅱ collagen and enhanced toluidine blue staining. And the transcription of type Ⅱ collagen and aggrecan genes was also increased,especially in the ADSC cultured in fibrin scaffold with 10% FBS. ［Conclusion