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目的探讨龟板提取物诱导大鼠骨髓间充质干细胞(mesenchymalstem cells,MSCs)向成骨分化和对维生素D受体(VDR)表达的影响。方法从大鼠骨髓中分离MSCs,体外培养,利用流式细胞术检测MSCs表面抗原CD44细胞标志,体外培养MSCs分成不同的组观察其向成骨分化,在培养基中不加任何处理的作为对照组,培养基中加成骨诱导液诱导MSCs向成骨分化作为阳性对照组,龟板提取物组在培养基中加龟板提取物,诱导7d后,通过免疫化学染色、Western blotting、原位杂交、RT-PCR等方法观察龟板提取物对原代培养的MSCs向成骨分化的成骨分化标记分子碱性磷酸酶(ALP)、骨桥蛋白(OPN)及VDR阳性表达及VDR mRNA的表达情况。结果免疫组化染色显示,龟板提取物组成骨分化标记分子ALP、OPN和VDR的阳性百分比明显高于对照组,Western blotting结果也显示龟板提取物组的ALP、OPN和VDR的蛋白表达水平高于对照组;同时原位杂交、RT-PCR结果表明,龟板提取物诱导MSCs向成骨分化过程可明显促进VDR mRNA的表达。结论龟板提取物可促MSCs向成骨分化,其机制可能与VDR的上调有关。
Objective To investigate the effects of turtle shell extract on osteogenic differentiation and vitamin D receptor (VDR) expression in rat mesenchymal stem cells (MSCs). Methods MSCs were isolated from rat bone marrow and cultured in vitro. Flow cytometry was used to detect the expression of CD44 on MSCs surface antigen. MSCs were cultured in vitro and observed for osteogenic differentiation without any treatment in the culture medium The osteogenic differentiation of MSCs induced by addition of osteogenic medium induced by addition of osteogenic medium was used as the positive control group. The turtle shell extract group was added with the turtle shell extract in the medium for 7 days. After induction for 7 days, the cells were harvested by immunochemical staining, Western blotting, RT-PCR and other methods were used to observe the expression of osteoblast differentiation marker alkaline phosphatase (ALP), osteopontin (OPN) and VDR and the expression of VDR mRNA of the cultured osteoblasts derived from turtle shell extract. Results Immunohistochemical staining showed that the positive percentage of ALP, OPN and VDR in turtle shell extract was significantly higher than that in control group. Western blotting also showed that the protein expression levels of ALP, OPN and VDR in turtle shell extract group were higher than those in control group The results of RT-PCR showed that the expression of VDR mRNA was significantly promoted by the induction of MSCs to osteogenic differentiation by the turtle shell extract. Conclusion The extract of turtle shell can promote the differentiation of MSCs into osteoblasts, which may be related to the up-regulation of VDR.