龟板提取物上调维生素D受体表达促骨髓间充质干细胞向成骨分化

Induction of Plastrum testudinis extract to mesenchymal stem cells differentiation into osteoblast through up-regulating expression of vitamin D receptor

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 vitamin D receptor （VDR） mechanism implicated in the osteoblast differentiation of mesenchymal stem cells （MSCs） induced by Plastrum testudinis extracts （PTE）. Methods MSCs were dissociated from rat bone marrow and were marked using the expression of CD44 by the flow cytometry （FCM）. MSCs Cultured in vitro were divided into different groups for the effects of MSCs differentiation into osteoblast, MSCs seeded in culture medium without treatment served as controls, MSCs seeded in culture medium with osteo-induction treatment served as positive controls, PTE was added and the cells were incubated for 7 d. Then immunohistochemistry, Western-blotting, in situ hybrid- ization, and RT-PCR were applied to observing the marker （ALP, OPN） as well as the VDR and VDR expression of the osteoblast differentiation specific mRNA expression. Results Immunohistorchemistry results demonstrated that the percentages of ALP, OPN, and VDR positive cells in the PTE group were markedly higher than those in the control group. The Western-blotting also illustrated that the expressing of ALP, OPN, and VDR in the PTE group was higher than that in the control group. In parallel, in situ hybridization and RT-PCR results showed that the expression of VDR reRAN was significantly increased during the osteoblast differentiation of MSCs induced by PTE. Conclusion The PTE could promote MSCs differentiation into osteoblast, which might be associated with the up-regulation of VDR.