共培养诱导人脐血间充质干细胞向软骨细胞分化*★

摘要

背景：研究发现共培养可诱导人源干细胞向特定细胞分化，但如何控制两种细胞间比例以最大效率获得目的细胞是研究中的难题。目的：观察人脐血间充质干细胞与兔软骨细胞按不同比例共培养后向软骨细胞分化的情况。方法：复苏人脐血间充质干细胞并进行传代，将经流式细胞仪鉴定的人脐血间充质干细胞与体外分离的兔软骨细胞按照2∶1或3∶1的比例共培养，并用100μg/L的胰岛素样生长因子1进行诱导。在共培养14 d，提取细胞RNA和蛋白质，实时定量PCR检测聚集蛋白多糖和Ⅱ型胶原mRNA的表达，Western blot检测聚集蛋白多糖和Ⅱ型胶原蛋白的表达。结果与结论：人脐血间充质干细胞与兔软骨细胞共培养14 d，共培养的细胞聚集蛋白多糖和Ⅱ型胶原mRNA及蛋白的表达高于单纯胰岛素样生长因子1诱导的细胞。在人脐血间充质干细胞与软骨细胞比例相同时，加入胰岛素样生长因子1可提高细胞聚集蛋白多糖和Ⅱ型胶原mRNA及蛋白的表达。同时人脐血间充质干细胞与兔软骨细胞按3∶1共培养更能促进细胞Ⅱ型胶原mRNA和蛋白及聚集蛋白多糖蛋白的表达；而按2∶1共培养时，细胞聚集蛋白多糖mRNA表达更多。可见人脐血间充质干细胞与兔软骨细胞共培养后可诱导人脐血间充质干细胞向软骨细胞分化，且按3∶1的比例共培养可获得更多的软骨细胞。%　　BACKGROUND: Human umbilical cord blood-derived mesenchymal stem cel s can be induced through the co-culture to differentiate into other cel s; however, it is difficult to control the proportions of two kinds of cel s for maximizing the efficiency of obtaining target cel s. OBJECTIVE: To investigate chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cel s by co-culture with rabbit chondrocytes at different ratios. METHODS: Human umbilical cord blood-derived mesenchymal stem cel s were resuscitated and cultured, and identified by flow cytometry. Then human umbilical cord blood-derived mesenchymal stem cel s were co-cultured with rabbit chondrocytes at 2:1 or 3:1 ratio, and induced with insulin-like growth factor-1. After 14 days in co-culture, the cel RNA and protein were extracted, aggrecan mRNA and col agen type Ⅱ mRNA expression levels were detected by real-time quantitative-polymerase chain reaction, and aggrecan and col agen type Ⅱ protein expression levels were detected by western blot assay. RESULTS AND CONCLUSION: After human umbilical cord blood-derived mesenchymal stem cel s were co-cultured with rabbit chondrocytes for 14 days, the aggrecan mRNA and col agen type 2 mRNA expression in the co-cultured cel s was significantly higher than that in the cel s only induced with insulin-like growth factor-1. Under the same ratio of human umbilical cord blood-derived mesenchymal stem cel s and rabbit chondrocytes, insulin-like growth factor-1 increased the aggrecan and col agen type Ⅱ protein and mRNA expressions. A 3:1 co-culture ratio al owed the increment in the col agen type Ⅱ mRNA and protein expression, as wel as aggrecan protein expression; while a 2:1 co-culture ratio upregulated aggrecan mRNA expression. Experimental findings indicate that, human chondrocytes can be successful y induced by co-culture of human umbilical cord blood-derived mesenchymal stem cel s and rabbit chondrocytes, and 3:1 co-culture ratio can obtain more chondrocytes.