体外共培养诱导脂肪干细胞向软骨表型细胞分化及鉴定的实验研究

The study on inducing adipose-derived stem cells into chondrogenic phenotype cells by co-culture and identification in vitro

目的:探讨脂肪干细胞经共培养诱导为软骨表型细胞的可行性,为软骨组织工程种子来源提供的新途径。方法:分离培养日本大耳白兔的脂肪干细胞及肋软骨细胞,将脂肪干细胞与软骨细胞分层共培养及脂肪干细胞单独培养于6孔板中2周。通过safranin-O染色、甲苯胺蓝染色及Ⅱ型胶原免疫化学染色对诱导后细胞表型进行鉴定,RT-PCR检测诱导后Ⅱ型胶原基因表达情况。结果:共培养诱导2周后的脂肪干细safranin-O染色和甲苯胺蓝染色及Ⅱ型胶原免疫化学染色呈阳性,Ⅱ型胶原基因表达接近于正常软骨细胞。结论:脂肪干细胞经与软骨细胞共培养后,可以诱导为软骨表型细胞。

Objective：To investigate the feasibility of inducing adipose-derived stem cells（ADSC） into chondrogenic phenotype cells by co-culture,in order to provide new approaches of seed cells sources for cartilage tissue engineering. Methods： Isolate and culture the Japanese white rabbits＇ adipose-derived stem cells and costal cartilage cells. ADSC and chondrocytes were co-cultured under new- style layer, and ADSC were solo-cultured in 6-well plates for 2 weeks. The phenotype was identified with the safranin O staining,tolui- ding blue staining and collagen II immunocytochemistry staining,and expression levels of collagen II were detected by PT-PCR. Re- sults： The safranin O staining, toluidine blue staining and collagen II immunoeytochemistry staining of the ADSC after 2 weeks co-cul- ture inducing were positive,and the expression levels of collagen III were close to the normal chondrocytes. Conclusion： Adipose-de- rived stem cells could be induced into chondrogenic phenotype cells by co-culture with chondrocytes.