胶原海绵上诱导骨髓间充质干细胞向软骨细胞分化的体外实验研究

In Vitro Study of the Directed Inducing Cartilage Cells from Rat Bone Mesenchymal Stem Cells on Scaffolds

研究在胶原海绵上间充质干细胞(M SC s)向软骨细胞的定向诱导,以及其对M SC s细胞增殖力、细胞周期的影响,为临床应用胶原海绵作为M SC s支架、修复软骨缺损奠定基础。取大鼠股骨骨髓,经梯度离心法和贴壁法分离M SC s,用加有转化生长因子1β10 ng/m l、地塞米松1-0 7m o l/L、转铁蛋白3 m g/m l、胰岛素2 m g/m l的诱导因子培养基分别在胶原海绵、培养板中进行诱导,14 d后免疫组化检测II型胶原分泌情况,四甲基偶氮唑蓝(M TT)法比较两者之间细胞增殖情况,流式细胞仪(FCM)检测各组细胞周期。14 d后,胶原海绵上及培养板中均诱导出表达II型胶原的软骨细胞,M TT法检测胶原海绵诱导组,和培养板诱导组细胞增殖值分别为0.9213±0.0312和0.5875±0.0258,两者间有显著性差异(P<0.05),FCM检测胶原海绵各组S期+G2/M期百分率,发现其与培养板中同期细胞相比有显著性差异(P<0.05),各组均未发现异倍体。结果表明在胶原海绵上,M SC s能定向诱导为软骨细胞,细胞增值能力更强,M SC s细胞DNA合成、细胞分裂增加,无异倍体产生,可作为一种较为理想的组织工程学支架。

This study was conducted to assess the cell proliferation and cell cycle of the directed inducing cartilage cells from rat hone mesenchymal stem cells on collagen sponge and compare the results with those of the plane inducing cartilage cells. The MSCs were isolated from rat bone marrow by density gradient centrifuge and adhesive property, and then were induced into chondrogenic differentiation by adding transforming growth factor-β1 （TGF-β1）, dexamethasone , transferring and insulin on collage sponge and plane condition. Immunohistochemistry was applied to detect the expression of collagen type II on the scaffolds and plane conditions. The cell proliferation and cell cycle on different conditions were detected with MTT automated colormetric microassay and flow cytometry. The differentiation of MSCs toward chondrogenic phenotype was verified by the positive result of collagen type Ⅱ on the scaffolds and plane condition. The cell viability in scaffolds was better than that in plane conditions. The S phase and G2 phase were 30 percent in whole cell cycle on the scaffold ,which was different from that of the control groups. The method of using the Using directed inducing cartilage from bone MSCs combined with collage sponge to construct tissue-engineered cartilage is feasible; it is better than the method of plane directed inducing.