体外诱导骨髓间质干细胞向软骨方向分化的研究

In vitro differentiation of rabbit bone marrow-derived mesenchymal stem cells into condrocytes

目的 探讨体外诱导骨髓间充质干细胞 (BMSCs)向软骨方向分化的条件和方法。方法 抽取兔股骨骨髓 ,用密度梯度离心和贴壁分离法分离BMSCs ,分别在高密度 (1× 10 6/ml)和低密度 (1× 10 4/ml)培养条件下用转化生长因子 β1(TGF β1)诱导BMSCs向软骨方向分化。倒置显微镜、透射及扫描电子显微镜观察细胞 ,用免疫组织化学、原位杂交、特殊染色方法检测Ⅱ型胶原及软骨基质成分的表达。结果 高密度诱导的BMSCsⅡ型胶原免疫组织化学阳性率为 89.2 %,Ⅱ型胶原原位杂交阳性率为 82 .6%,黏多糖特殊染色阳性 ,低密度诱导的BMSCsⅡ型胶原免疫组织化学阴性 ,Ⅱ型胶原原位杂交阳性率为 3 .8%,黏多糖特殊染色阴性。结论 TGF β1可以诱导BMSCs向软骨方向分化 ,细胞密度是BMSCs分化的重要影响因素。

ObjectiveTo establish a system for inducing differetiation of rabbit bone marrow-dirived mesenchymal stem cells (BMSCs) into chondrocytes.MethodsBMSCs were isolated from rabbit bone marrow aspirates by density gradient centrifugation and adhere culture.TGF-β1 was used to induce chondrogenesis differentiation of BMSCs at high density culture (1×10 6/ml) and low density culture (1×10 4/ml).Cells were observed under invert microscopy and electron microscopies.Collagen II were detected by immunohistochemistry and in situ hybridization,and GAG was detected by toluidine blue and AB-PAS.ResultsThe positive rate of Collagen II immunohistochemistry and in situ hybridization induced by TGF-β1 in highy density of BMSCs was 82.6% and 3.8% respectively.BMSCs in low density culture didn't express chondrocyte phenotype until 14 days.ConclusionTGF-β1 can induce the chondrogenesis differetiation of BMSCs.The cell density is an important factor in the chondrogenesis differetiation of BMSCs.