Transwell小室环境下兔成骨样细胞与骨髓基质干细胞的共培养及成骨分化

Co-cultivation of bone marrow stromal cells with rabbit osteoblast-like cells in a Transwell chamber for osteogenic differentiation

背景:以往在体外采用地塞米松、生长因子或用成骨样细胞与骨髓间充质干细胞按1∶1混合培养诱导成骨均存在种种局限。目的:观察在 Transwell小室环境下成骨样细胞与骨髓基质干细胞体外共培养及成骨样细胞定向诱导骨髓基质干细胞向成骨细胞分化的可行性。方法:取第 3代乳兔成骨样细胞与第3代兔骨髓基质干细胞接种共培养于Transwell小室内,成骨样细胞接种于培养板底层,骨髓基质干细胞接种于 Transwell膜内膜上作为实验组。以骨髓基质干细胞单独接种于Transwell 小室内,下层为基础培养液作为对照组。结果与结论:实验组共培养骨髓基质干细胞明显向成骨细胞分化,细胞碱性磷酸酶活性显著高于对照组(P < 0.05)。实验组骨髓基质干细胞茜素红染色强阳性,可见呈红色结节,经PT-PCR扩增后,可见成骨启动基因核心结合因子α1的表达;对照组未见矿化结节。说明应用Transwell小室可实现成骨样细胞与骨髓基质干细胞体外共培养,并能定向诱导骨髓基质干细胞向成骨细胞分化。

BACKGROUND： Osteogenic differentiation is previously limited by culture of dexamethasone, growth factors or osteoblast-like cells with bone marrow mesenchymal stem cells at 1：1. OBJECTIVE： To investigate the feasibility of in vitro co-cultivation of osteoblast-ike cells and bone marrow stromal cells （BMSCs） and osteoblast differentiation of BMSCs by osteoblast-like cells. METHODS： Passage 3 rabbit osteoblast-like cells and passage 3 rabbit BMSCs were co-cultured in Transwell chamber, and osteoblast-like cells were inoculated into the lower part of culture plate. BMSCs were inoculated on Transwell inner membrane, serving as experimental group. BMSCs were inoculated into Transwell chamber alone with basal medium in the lower part, serving as control group. RESULTS AND CONCLUSION： In the experimental group, BMSCs differentiated toward osteoblasts, and alkaline phosphatase activity was significantly higher compared with the control group （P 〈 0.05）. In the experimental group, BMSCs were positive for alizarin red staining, showing red calcified nodules; after RT-PCR, the expression of core-binding factor α1 was observed. In the control group, calcified nodules were not observed. These findings suggest that Transwell chamber can help to achieve the in vitro co-cultivation of osteoblast-like cells and BMSCs and the osteogenic differentiation of BMSCs.