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背景:骨髓基质细胞一定条件下可以分化为软骨细胞,但目前还缺少将软骨与骨髓基质细胞平面共培养的深入探讨。目的:拟明确骨髓基质细胞与软骨细胞共培养时比例的选择、细胞增殖活性、软骨特异性蛋白表达的规律,用于细胞移植的最佳时间和传代次数。方法:软骨细胞与骨髓基质细胞的分离培养,传代后分为5组:单纯软骨细胞组;共培养组,软骨细胞与骨髓基质细胞7:3,5:5,3:7组;以及单纯骨髓基质细胞组。传代至第4代(G4),倒置相差显微镜观察各组细胞在不同传代时期的细胞形态,MTT实验检测细胞增殖活性,甲苯胺蓝与阿利新蓝染色,细胞免疫化学法检测Ⅱ型胶原的表达。结果与结论:共培养各组在各个时期细胞表型正常。G1~G3代细胞增殖活跃,G4代细胞增殖能力下降。共培养各组甲苯胺蓝与阿利新蓝染色以及Ⅱ型胶原表达在G2和G3代均较高。综合各指标认为,以软骨细胞与骨髓基质细胞的比例为5:5,3:7组为最佳。软骨细胞与骨髓基质细胞平面共培养,保持了软骨细胞的形态和蛋白表达特性,如进行细胞移植,选取软骨细胞与骨髓基质细胞5:5或3:7的比例为最佳。
BACKGROUND: Bone marrow stromal cells can differentiate into chondrocytes under certain conditions. However, there is still a lack of discussion on the co-culture of cartilage and bone marrow stromal cells. OBJECTIVE: To determine the proportion of bone marrow stromal cells co-cultured with chondrocytes, the cell proliferation activity, the expression of cartilage-specific proteins, the optimal time for cell transplantation and the number of passages. Methods: Chondrocytes and bone marrow stromal cells were isolated and cultured. After passage, they were divided into 5 groups: pure chondrocyte group, co-culture group, chondrocytes and bone marrow stromal cells in 7: 3, 5: 5 and 3: Stromal cell group. The cells were passaged to passage 4 (G4). The morphological changes of cells in different passage passages were observed by inverted phase contrast microscope. MTT assay was used to detect the cell proliferative activity. Toluidine blue and alitame blue staining were used to detect the expression of type Ⅱ collagen . RESULTS AND CONCLUSION: The co-cultured cells showed normal cell phenotype at all stages. G1 ~ G3 generation of cell proliferation, G4 generation cell proliferation decreased. Toluidine blue and alitame blue staining and type II collagen expression in co-cultured groups were higher in G2 and G3 generation. Comprehensive indicators that the ratio of chondrocytes and bone marrow stromal cells 5: 5,3: 7 group is the best. Chondrocytes are co-cultured with bone marrow stromal cells in a planar manner to maintain the morphology and protein expression characteristics of chondrocytes. For example, the best ratio of chondrocytes to bone marrow stromal cells is 5: 5 or 3: 7.