骨髓间充质干细胞体外诱导成软骨细胞的动态观察

背景：骨髓间充质干细胞向软骨细胞定向诱导时间报道不一,对诱导中细胞动态变化进行观察比较的研究较少。目的：观察兔骨髓间充质干细胞诱导8,11,14,17,20 d向成软骨细胞定向分化的动态变化情况以及诱导成熟时间。方法：穿刺新西兰大白兔股骨穿刺抽取骨髓,密度梯度离心法分离、培养骨髓间充质干细胞。传至第3代后予以含转化生长因子β1等成分的无血清诱导液定向向软骨细胞诱导。根据不同的诱导时间分成为5组：8 d组、11 d组、14 d组、17 d组及20 d组。通过观察各组细胞形态、甲苯胺蓝染色结果、Ⅱ型胶原免疫组化结果、培养液内多聚蛋白聚糖含量,比较各实验组定向诱导为成软骨细胞的情况。结果与结论：诱导8 d时细胞形态有较明显改变,14 d时具有明显的软骨细胞形态。诱导培养液在诱导4 d时即可检出少量多聚蛋白聚糖,在8 d时浓度出现明显升高,并保持缓慢升高至20 d。在14 d时,诱导的细胞爬片甲苯胺蓝染色可见异染颗粒;Ⅱ型胶原免疫组化明显阳性表现。提示单层高密度接种的骨髓间充质干细胞,在转化生长因子β1等因子作用下能定向诱导为软骨细胞,诱导早期即可能有少量细胞向成软骨细胞分化,8 d时初步具有软骨细胞的形态、功能特征,14 d时成为较成熟的软骨细胞;诱导过程中细胞保持较高的生物活性。

Dynamically observing chondrogenic differentiation of bone marrow mesenchymal stem cells in vitro

BACKGROUND: The reported time of bone marrow mesenchymal stem cells induced to differentiate into chondrocytes is different. Few studies have observed and compared the cells’ dynamic transformation during the induction process.OBJECTIVE: To observe the dynamic differentiation and the mature time of rabbit bone marrow mesenchymal stem cells which were directionally induced to chondroblasts for 8, 11, 14, 17, 20 days.METHODS: Bone marrow was aspirated from the femur of New Zeal rabbits, and bone marrow mesenchymal stem cells were isolated by gradient centrifugation. After cultivation and amplification, bone marrow mesenchymal stem cells at passage 3 were directionally induced to chondrocytes by the serum-free medium containing transforming growth factor beta-1. The experiments were divided into five groups according to different induction time points: 8 days, 11 days, 14 days, 17 days, 20 days. Then cellular morphology, toluidine blue staining, type Ⅱ collagen immunohistochemistry, aggrecan content in induction medium, and chondrogenic differentiation in each group were observed and compared.RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells had apparently transformed in morphology at 8 days of induction, and presented obvious chondrocytes’ morphology at 14 days. The aggrecan in induction medium could be detected at a low level at 4 days, significantly increased at 8 days, and maintained slow increasing at 20 days. At 14 days, the metachromatic particles could be found by toluidine blue staining, and the collagen type Ⅱ immunohistochemistry was significantly positive in cell climbing slice. Experimental findings indicate that, bone marrow mesenchymal stem cells that are monolayer cultured in a high density can be induced into chondroblasts at the effect of transforming growth factor beta-1 and other factors. There are a few chondroblasts in the early induction process, then cells begin to have chondrocytes morphology and function after induced for 8 days, and may differentiate to mature chondrocytes at 14 days. In addition, they can keep a high biological activity in the induction process.