兔骨髓间充质干细胞体外软骨向诱导的实验研究

目的:体外分离培养兔骨髓间充质干细胞(MSCs),并定向诱导其向软骨细胞表型分化。方法:抽取兔股骨骨髓,经密度梯度离心与贴壁培养分离得到MSCs。用含有TGF-β、b-FGF、胰岛素、维生素C、转铁蛋白等的DMEM/Ham′s-F12培养基进行软骨向诱导。通过形态学观察,阿辛蓝-PAS特染及Ⅱ型胶原免疫组化染色,鉴定经过诱导后细胞的软骨细胞的表型。结果:兔MSCs原代细胞呈梭形,克隆样生长,经过诱导培养7d后,细胞形态由梭形、多角形变为椭圆形,胞浆丰富,细胞核和核仁清晰可见。阿辛蓝-PAS和Ⅱ型胶原染色阳性。结论:密度梯度离心与贴壁培养两种方法相结合可获得相对高纯度的MSCs,该细胞经诱导后可向软骨细胞分化。

Experimental study of chondrogenic induction of rabbit bone marrow mesenchymal stem cells in vitro

Objective:To isolate and culture the rabbit bone marrow mesenchymal stem cells (MSCs)for inducing them directly into phenotypic chondrocytes in vitro. Methods: The marrow was extracted from femoral bone of New Zealand white rabbit for isolating MSCs by means of density gradient centrifugation and adherent culture. After cells induction by DMEM/Ham′s-F12 medium containing TGF-β, b-FGF, insulin, VitC and transferrin, cell morphological observation, alcian blue-PAS histochemistry and collagen type Ⅱ immuno-histochemistry were conducted for chondrogenic phenotype identification. Results: Primary MSCs proliferated in visible colonies with spindle shape. After 7 days of induction, some of the MSCs developed from spindle-shaped or polygon to elliptic shape with abundant endochylema, clear nucleus and nucleoli, showing positive staining of alcian blue and collagen type Ⅱ. Conclusion :Combination approach of density gradient centrifugation and adherent culture may generate highly purified MSCs, and the isolated MSCs can differentiate into cartilage cell under special culture condition.