两种培养体系条件下人脐带间充质干细胞向软骨细胞的分化

Induced differentiation of human umbilical cord mesenchymal stem cells into chondrocytes in two kinds of culture systems

背景:无支架体外诱导间充质干细胞向软骨细胞分化的方法主要包括高密度微团培养、单层细胞培养、与软骨细胞体外共培养等,其中高密度微团培养和体外单层细胞培养因操作简便易行,诱导成功率高受到广泛关注。目的:寻求一种更佳的无支架体外诱导人脐带间充质干细胞向软骨细胞分化的方法。方法:组织块法分离人脐带间充质干细胞,流式细胞仪鉴定其表面标志。收集第3代人脐带间充质干细胞分别采用平面诱导培养和离心管聚集成团培养,使其向软骨细胞分化。结果与结论:人脐带间充质干细胞的细胞表型CD105+/CD29+/CD44+/CD31-/CD34-/CD40-CD45-/HLA-DR-。未诱导的人脐带间充质干细胞表达微弱的软骨细胞标志物,经诱导后葡萄糖胺聚糖和Ⅱ型胶原表达量显著增加,其中聚集成团培养的表达量高于平面培养。实时定量PCR结果显示,诱导后细胞较诱导前均高表达葡萄糖胺聚糖、Ⅱ型胶原和SOX-9mRNA,其中聚集成团培养的表达量高于平面培养。说明人脐带间充质干细胞在聚集成团培养体系中诱导更有利于其分化成软骨细胞。

BACKGROUND：The methods of inducing mesenchymal stem cells to differentiate into chondrocytes under non-scaffold condition include high-density pellet culture,in vitro monolayer culture and co-culture with chondrocytes.High-density pellet culture and in vitro monolayer culture are widely concerned due to simple operation.OBJECTIVE：To find a better method of inducing human umbilical cord mesenchymal stem cells into chondrocytes in non-scaffold culture in vitro.METHODS：Human umbilical cord mesenchymal stem cells were isolated and cultured by tissue block method,and cell surface antigens were identified by flow cytometry.Human umbilical cord mesenchymal stem cells were cultured with chondrogenic media and stained with alcian blue.The production of matrix was estimated from the determination of hydroxyproline content using alcian blue method.Expressions of glycosaminoglycan,type Ⅱ collagen and Sox-9 were assayed by real-time fluorescence quantitative PCR.RESULTS AND CONCLUSION：The phenotype of cultured human umbilical cord mesenchymal stem cells was CD105＋/CD29＋/CD44＋/CD31-/CD34-/CD40-/CD45-/HLA-DR-.Human umbilical cord mesenchymal stem cells weakly expressed chondrocyte markers,which strongly expressed glycosaminoglycan and type Ⅱ collagen after chondgenic induction,and the expressions were higher in cells cultured in pellet culture system than in monolayer culture system.Real-time quantitative PCR results demonstrated that cells expressed higher level of glycosaminoglycan,type Ⅱ collagen and Sox-9 after induction than that before induction,and the expression levels were higher in cells cultured in pellet culture system than in monolayer culture system.The results revealed that human umbilical cord mesenchymal stem cells cultured in pellet culture system are more conducive to differentiate into chondrocytes than those cultured in monolayer culture system