不同来源软骨细胞共培养对骨髓间充质干细胞早期分化的影响

Influence of chondrocytes of different origin on early differentiation of bone marrow mesenchymal stem cells in co-culture system

目的了解不同来源的软骨细胞在细胞隔离共培养的环境中，对相同来源的骨髓间充质干细胞（MSCs）早期向软骨系分化的影响。方法利用悬挂式细胞培养皿，将不同来源的软骨细胞（骨性关节炎患者的软骨细胞、正常成年人的软骨细胞、胎儿软骨细胞）及对照MSCs，和同一来源的MSCs隔离培养在同一培养液中，共享细胞培养液，分别在培养3d、6d、9d、12d通过显微镜观察，并应用Real-timePCR技术测定经不同来源软骨细胞诱导的MSCs表达蛋白聚糖，Ⅱ型胶原（COL-2），软骨转录调控因子（SOX-9）信号的水平。结果MSCs在不同来源的软骨细胞诱导下，在细胞培养12d后，与对照组比较，出现了形态的明显区别。SOX-9的mRNA表达分别为对照组的1．7倍、1．6倍、1．2倍（P〈0．05）；蛋白聚糖的mRNA表达分别为对照组的2．8倍、2．2倍、1．3倍（P〈0．05），COL2的mRNA表达变化不大；相应蛋白的信号水平变化表达也有较大差异，胎儿软骨细胞组相对于骨性关节炎患者的软骨细胞组及正常成年人的软骨细胞组更加明显。结论隔离共培养体系能够通过局部微环境的调节，具有间接促进MSCs定向诱导分化成软骨细胞的作用，不同来源的软骨细胞促分化的影响不同，但都具有促MSCs定向诱导分化成软骨细胞的能力。

Objective To investigate the influence of chondrocytes originating from different source on early ehondrogenic differentiation of bone marrow mesenehymal stem cells （MSCs） in isolated co-culture system. Methods We applied hanging cell-culture system to culture chondrocytes of different origin （osteoarthritis chondroeyte cells, nomal chondrocyte cells, infant chondrocyte cells） and controls. These chondrocytes and MSCs of the same origin were cultured in the common medium in a separated condition, and observed by microscope at 3, 6, 9, 12 day after co- culture. Expression levels of aggrecan, collagen type lI （Col-2）, cartilage-specific transcription factor （Sox-9） in MSCs of different origin were determined by Real-time PCR. Results MSCs showed obviously morphological differentiation induced by chondrocytes of different origin at 12 day after co- culture as compared with controls. Real-time PCR analysis showed that SOX9 mRNA level was stimulated by 1.7-fold, 1.6-fold and 1.2-fold （all P^0.05） and aggrecan mRNA level was increased by 2.8-fold, 2.2-fold and 1.3-fold （all P^0.05） in infant chondrocytes group, nomal chondrocytes group, osteoarthritis chondrocytes group respectively as compared with controls while COL2 mRNA level had no significant differences among the four groups. Corresponding protein signal level had obvious differences among the four groups, especially in infant chondrocytes as compared with osteoarthritis chondrocytes and nomal chondrocytes. Conclusions Isolated co-culture system may indirectly promote MSCs differentiation to chondrocytes by local micro-environment regulation.Chondrocytes of different origin have different effects on MSCs differentiation, but they could promote MSCs differentiation to chondrocytes.