间充质干细胞对大鼠退变软骨细胞的作用

Effects of mesenchymal stem cells on degenerative chondrocytes in rats

目的探讨间充质干细胞(MSCs)对大鼠退变软骨细胞的作用。方法取SD大鼠骨髓,分离单核细胞,贴壁培养,用诱导培养基对细胞进行定向诱导。采用油红O、茜素红和番红O染色检测定向诱导效果。第3代MSCs种植在Transwell膜上,第2代软骨细胞种植在6孔板底部。IL-1β诱导后,收集单独培养和与MSCs共培养3d的软骨细胞,采用RT-PCR及Western blot法分别检测Ⅱ型胶原(Col2)、蛋白聚糖mRNA和蛋白的表达,同时收集单独培养及与MSCs共培养1d的软骨细胞检测环氧合酶2(COX-2)、MMP-13mRNA和蛋白的表达。结果所得到的MSCs形态均一,呈长梭形,类似成纤维细胞的形态。经呈脂肪、成骨、软骨细胞诱导培养后,油红O、茜素红及番红O染色显示MSCs有向以上3个方向诱导分化的特性。MSCs抑制IL-1β诱导的大鼠软骨细胞内MMP-13和COX-2的表达,增加Col2和蛋白聚糖的表达。结论 MSCs通过下调退变软骨细胞中MMP-13和COX-2的表达,抑制了Col2和蛋白聚糖的降解,对大鼠退变软骨细胞有明显改善作用。

Objective To investigate the effects of mesenchymal stem cells （MSCs） on the degenerative chondrocytes in rats. Methods Fresh bone marrow of SD rats was extracted from the tibal and femur, and MSCS were isolated by density gradient centrifugation through Percoll. The cells were induced directionally to adipogenic, osteogenic and chondrogenic lineages. Confluent rat chondrocytes were cultured indirectly with or without MSCs at a ratio of 2 ： 1, and then treated with IL-1β 10 ng/ml. Total RNA and protein were collected at different incubation times. Western blot and RT-PCR were used to detect the expressions of type II collagen（Col2）, aggrecan, matrix metalloproteinase-13（MMP-13） and cyclooxygenase-2 （COX-2）. Results The MSCs were induced to adipogenic,osteogenic and chondrogenic lineages successfully, which enhanced the expressions of Col2 and aggrecan, and inhibited the expressions of MMP-13 and COX-2 in IL-1β-stimulated rat chondrocytes. Conclusion The MSCs suppress the degradation of Col2 and aggrecan through down- regulating the expressions of MMP-13 and Col2 in the degenerative chondrocytes induced by IL-1β, which benefits the improvement of the degenerative chondrocytes.