左归丸含药血清调节Runx2激活ALP、OPN蛋白促进BMSCs增殖和分化的研究

Study on the promotion of proliferation and differentiation of BMSCs by Runx2 activation of ALP and OPN proteins with Zuoguiwan containing serum

目的探讨左归丸(Zuoguiwan,ZGW)的补肾填精、益髓壮骨的作用,促进骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的增殖和向成骨细胞方向分化。方法制备ZGW含药血清,设置10%FBS DMEM组、成骨诱导液组、2.5%空白大鼠血清-成骨诱导液组、2.5%ZGW含药血清-成骨诱导液组。采用MTT法观察各组对BMSCs的增殖,使用碱性磷酸酶钙钴染色法检测BMSCs向成骨细胞方向的分化能力,碱性磷酸酶(alkaline phosphatase,AKP)法检测细胞培养液中AKP的含量,Western blot检测BMSCs中Runt相关转录因子2(Runx2)、骨钙蛋白(osteocalcin,OPN)和碱性磷酸酶(alkaline phosphatase,ALP)蛋白的表达。结果2.5%ZGW含药血清-成骨诱导液组能够促进BMSCs的快速增殖,较早地进入平台期,促进BMSCs中AKP的分泌,提高ALP含量,上调BMSCs细胞中Runx2、ALP和OPN的蛋白表达水平。结论ZGW抗骨质疏松的分子机制,可能与调节Runx2激活ALP、OPN蛋白表达,促进BMSCs增殖和向成骨细胞方向分化有关。

Objective To study the effects of Zuoguiwan(ZGW)on nourishing kidney essence,strengthening bone and marrow,and promotion of the proliferation of bone marrow mesenchymal stem cells(BMSCs)and differentiation into osteoblasts.MethodsThe ZGW containing serum was prepared.10%FBS DMEM group,osteogenic liquid group,2.5%serum of health rats with osteogenic liquid group,and 2.5%serum containing ZGW osteogenic liquid group were set up.MTT method was used to observe the proliferation of BMSCs in each group.The differentiation of BMSCs into osteoblasts was tested using alkaline phosphatase calcium cobalt staining method.Alkaline phosphatase(AKP)method was used to detect the content of AKP in cell culture medium.The expression of runt related transcription factor 2(Runx2),osteocalcin(OPN),and alkaline phosphatase(ALP)protein in BMSCs were detected using Western blotting.Results Rapid proliferation of BMSCs was showed in 2.5%serum containing ZGW osteogenic liquid group.The plateau stage appeared earlier.It promoted the secretion of AKP in BMSCs,increased the content of ALP,and up-regulated the protein expression levels of Runx2,ALP,and OPN in BMSCs cells.Conclusion The molecular mechanism of ZGW against osteoporosis may be related to the regulation of Runx2,activation of ALP and OPN protein expression,promotion of BMSCs proliferation and differentiation into osteoblasts.