骨髓间充质干细胞向软骨细胞的分化

背景：以骨髓间充质干细胞构建组织工程气管尚缺乏理想的特异性表面标志物，对其鉴定主要依赖细胞形态学、细胞表型及诱导分化的功能进行分析。目的：体外分离培养、鉴定兔骨髓间充质干细胞，观察在特定条件下向气管软骨细胞分化的潜能。方法：无菌环境取兔骨髓，经全骨髓贴壁筛选法分离培养细胞至第2代，流式细胞术鉴定第1、第2代细胞表面抗原CD44、CD45的表达。无菌环境取气管，经酶消化法分离培养气管软骨细胞，甲苯胺蓝染色鉴定软骨细胞蛋白聚糖的合成。在使用转化生长因子B1的基础上，将骨髓间充质干细胞与气管软骨细胞通过Transwell小室非接触式共培养，倒置显微镜观察细胞形态，甲苯胺蓝染色鉴定蛋白聚糖的合成，荧光实时定量PCR鉴定Ⅱ型胶原和蛋白聚糖mRNA的表达。结果与结论：分离、培养的细胞呈长梭形、不规则形聚集生长，传代后细胞生长速度明显增快，呈鱼群状聚集生长。第1代有96．97％的细胞表达CD44、13.72％的细胞表达CD45，第2代有99．11％的细胞表达CD44、8．54％的细胞表达CD45。气管软骨细胞甲苯胺蓝染色阳性。在诱导后，骨髓间充质干细胞形态逐渐由长梭形变为三角形或不规则形，表达软骨细胞特异性Ⅱ型胶原和蛋白聚糖mRNA基因，甲苯胺蓝染色示阳性。结果表明全骨髓贴壁筛选法可成功分离培养骨髓间充质干细胞，第2代纯度较高，且在特定诱导条件下具有分化为气管软骨细胞的潜能。

Chondrogenic differentiation of bone marrow mesenchymal stem cells

Rabbit bone marrow was acquired in the sterile environment to isolate and culture bone marrow mesenchymal stem cells to passage 2 by bone marrow adherence and screening method. Flow cytometry identified the phenotype CD44, CD45 of bone marrow mesenchymal stem cells at passages 1 and 2. Rabbit tracheal samples were acquired in the sterile environment, the tracheal chondrocytes were isolated and cultured by enzyme digestion, and toluidine blue staining was used to detect aggrecan. Bone marrow mesenchymal stem cells were co-cultured w!th tracheal chondrocytes by Transwell and transforming growth factor 131. Cell morphology was detected under an inverted microscope. Real-time quantitative PCR and toluidine blue staining detected the extracellular matrix components, such as type I collagen and aggrecan.RESULTS AND CONCLUSION： After isolation and culture, cells were spindle and irregular in morphology, and passaged cells thrived that were gathered into a fish-like colony growth. For passage 1 bone marrow mesenchymal stem cells, the positive rates of phenotype antigen CD44 and CD45 were respectively 96.97% and 13.72%; for passage 2 cells the positive rates of phenotype antigen CD44 and CD45 were 99.11% and 8.54%, respectively. Tracheal chondrocytes were positive for toluidine blue staining. The morphology of induced bone marrow mesenchymal stem cells changed from long fusiform to triangular or irregular shape, indicating the chondrocytes expressed type Ⅱ collagen and aggrecan, and toluidine blue staining was positive. These results showed bone marrow adherence and screening method could acquire bone marrow mesenchymal stem cells, and the purity of passage 2 cells is higher. Under a special condition, bone marrow mesenchymal stem cells have the potential of chondrogenic differentiation, and can be selected as seed cells for construction of tissue-engineered trachea.