补肾活血汤水提物调控Cbfal/RUNX2基因沉默骨髓间充质干细胞SP7/Osterix及碱性磷酸酶的表达

Expression of SP7/Osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen Huoxue Decoction

背景:补肾活血汤可以促进骨髓间充质干细胞成骨分化,探究分子机制利于推向临床。目的:探讨补肾活血汤水提物对Cbfal/RUNX2基因沉默的骨髓间充质干细胞Osterix、碱性磷酸酶表达的影响。方法:骨髓贴壁筛选法分离和培养大鼠骨髓间充质干细胞,Cbfal/RUNX2基因沉默慢病毒转染骨髓间充质干细胞。取第3代骨髓间充质干细胞(空白对照组)、慢病毒沉默骨髓间充质干细胞(沉默组)和阴性病毒载体预处理骨髓间充质干细胞(阴性对照组),以及上述3组骨髓间充质干细胞加入100 mg/L补肾活血汤水提物干预3 d后,检测RUNX2、Osterix蛋白和mRNA表达以及碱性磷酸酶活性。结果与结论:(1)Cbfal/RUNX2基因沉默慢病毒转染效率达约90%;(2)与空白对照组和阴性对照组比较,沉默组骨髓间充质干细胞RUNX2和Osterix的蛋白及mRNA表达均下降,碱性磷酸酶活性也明显下降,差异有显著性意义(P<0.01);(3)补肾活血汤水提物干预后,沉默组骨髓间充质干细胞RUNX2、Osterix的表达和碱性磷酸酶活性明显增加,差异有显著性意义(P<0.01);(4)结果表明,补肾活血汤水提物可以促进骨髓间充质干细胞的RUNX2、Osterix表达,亦增加了碱性磷酸酶活性,从而促进骨髓间充质干细胞成骨分化。

BACKGROUND：Bushen Huoxue Decoction（BSHXD）can promote osteogenesis of bone marrow mesenchymal stem cells（BMSCs）in vitro.Exploring the molecular mechanisms involved is of clinical benefits. OBJECTIVE： To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase （ALP） in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD. METHODS： BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing （blank control group）, Cbfal/RUNX2 gene silencing lentivirus （silencing group）, and negative viral vector （negative control group）, respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group. RESULTS AND CONCLUSION： The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity （P 〈 0.01）. After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly （P 〈 0.01）. To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation.