兔骨髓间充质干细胞培养及向成软骨细胞的诱导分化

背景：骨髓间充质干细胞没有理想的特异性表面标志物,对其鉴定尚无统一标准,大多数鉴定依赖于其形态水平、功能特征及培养过程中出现的分化表型来协助鉴定。目的：体外培养扩增、鉴定兔骨髓间充质干细胞,观察其诱导分化为成软骨细胞的可行性。方法：密度梯度离心法提取兔骨髓间充质干细胞,采用倒置显微镜进行形态学观察;用含体积分数为10%胎牛血清的α-MEM培养液培养细胞,每日定时进行细胞计数,观察细胞生长速度;采用流式细胞学方法分别检测原代及第3代细胞CD44、CD45、CD29的表达情况;取第3代生长良好的兔骨髓间充质干细胞,以特定培养液诱导（含体积分数为10%胎牛血清、转化生长因子110μg/L、胰岛素样生长因子Ⅰ110μg/L、转铁蛋白6.25mg/L、地塞米松10mmol/L、维生素C0.05mmol/L）,倒置显微镜下观察形态变化,免疫组织化学检测软骨特异性Ⅱ型胶原的表达。结果与结论：培养的细胞长梭形,呈成纤维细胞样生长,传代细胞生长迅速。流式结果表明,原代细胞有62.4%的细胞表达CD29,有60.3%的细胞表达CD44,有2.79%的细胞表达CD45,第3代有99.9%的细胞表达CD29,有99.6%的细胞表达CD44,有2.62%的细胞表达CD45。骨髓间充质干细胞诱导后体外扩增能力明显降低,诱导21d后形态变化比较显著,Ⅱ型胶原阳性细胞较多。结果显示应用密度梯度离心法可成功分离并扩增骨髓间充质干细胞,第3代细胞纯度较高,在适当的条件下,具有分化为成软骨细胞的潜能。

Culture and differentiation of rabbit bone marrow mesenchymal stem cells into chondrocytes

BACKGROUND：Bone marrow mesenchymal stem cells（BMSCs） do not have ideal specific surface markers,and there is no unified standard to identify BMSCs.Most identification depends on its morphological levels,functional characteristics and differentiational phenotype occurred during the culture.OBJECTIVE：To culture,amplify and identify rabbit BMSCs in vitro,and observe the feasibility of differentiation into chondrocytes.METHODS：Rabbit BMSCs were extracted through the density gradient centrifugation.Cell morphology was observed using the inverted microscope.Cells were cultured in α-MEM medium containing 10% fetal bovine serum,and cells were counted daily to observe cell growth speed.Expression of CD45,CD29,CD44 on P0 and P3 BMSCs was detected using flow cytometry.Well-grew rabbit BMSCs at P3 were obtained and induced using special media supplemented with 10% fetal bovine serum,transforming growth factor（TGF）-1 10 μg/L,insulin-like growth factor（IGF）-Ⅰ 110 μg/L,transferring 6.25 mg/L,dexamethasone 10 mmol/L and vitamin C 0.05mmol/L.Morphological changes were observed using the inverted microscope.Specificity of cartilage collagen type Ⅱ was detected using immunohistochemical method.RESULTS AND CONCLUSION：The cultured cells in the fusiform long,fibroblast growth of samples.The cells grow rapidly.Flow cytometry results showed that for the P0 cells about 62.4% of the cells expressed CD29,about 60.3% of the cells expressed CD44,2.79% of the cells expressed CD45,and for the P3 cells,about 99.9% of the cells expressed CD29,about 99.6% of the cells expressed CD44,2.62% of the cells expressed CD45.After the BMSCs in induction,the ability of expansion was decreased obviously,and the morphology changes after 21 days,and the collagen type Ⅱbecome dyeing.Results suggest that BMSCs can be successfully isolated and amplified by density gradient centrifugation.The third passage of BMSCs shows high purity.Under appropriate conditions,the BMSCs have the potential of differentiating itself into chondrocytes.