羌活鱼含药血清对大鼠骨髓间充质干细胞增殖及成骨分化的影响

Effects of serum containing Bing's giant salamander on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells

背景:羌活鱼具有促进骨折愈合的作用,但其具体的作用机制和其有效成分的研究目前尚不清楚。目的:观察不同浓度羌活鱼含药血清对体外培养大鼠骨髓间充质干细胞成骨分化的影响。方法:以羌活鱼研粉剂灌服大鼠制得羌活鱼含药血清,灌服等体积生理盐水制得对照血清;采用贴壁筛选法培养大鼠骨髓间充质干细胞,并以不同浓度(2.5%,5%,7.5%,10%)羌活鱼含药血清干预第3代大鼠骨髓间充质干细胞,通过MTT法检测细胞增殖情况;分别测定细胞碱性磷酸酶活性、钙盐沉积量、骨钙素、Ι型胶原表达量以及钙化结节数。结果与结论:5%和7.5%浓度含药血清可促进细胞增殖、成骨性分化,尤以5%含药血清最为明显,其碱性磷酸酶活性、钙盐沉积量、Ι型胶原含量、骨钙素含量和钙化结节数均显著高于空白对照组(P<0.01)。羌活鱼经口服后的代谢产物可刺激大鼠骨髓间充质干细胞增殖,并能促进其成骨性分化,推测其可能是羌活鱼续断接骨的作用机制之一。

BACKGROUND： Bing＇s giant salamander has the effect to enhance the fracture healing, but the action mechanism and active constituent are still unclear. OBJECTIVE： To investigate the effects of the serum of Bing＇s giant salamander on the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells （rBMSCs） by different concentrations. METHODS： Rats were given Bing＇s giant salamander by high, moderate and low concentration, respectively, and their serum was obtained after 7 days. The control serum was obtained by giving equal volume physiological saline. rBMSCs were obtained from Wistar rats and screened by the adhesive method. The serum was supplemented into the culture medium of third passage rBMSCs by different concentration （2.5%, 5%, 7.5%, 10%）. The proliferation of rBMSCs was analyzed by MTT reduction assay. The osteogenic differentiation markers including alkaline phosphatase （ALP） activity, calcium deposition, Collagen Ⅰ, osteocalcin and mineralized bone modulus were compared among the exposed groups and the control. RESULTS AND CONCLUSION： The values of A570 in serum groups containing 5% and 7.5% Bing＇s giant salamander were significantly higher than those of other concentrations and the control. 5% was stronger than 2.5% in significantly enhancing the osteogenic differentiation of rBMSCs, indicated by significantly improved ALP activity, calcium deposition, Collagen Ⅰ, osteocalcin and the number of mineralized bone nodules compared to the control and other groups （P 0.01）. The metabolites of Bing＇s giant salamander after oral administration stimulate the proliferation and osteogenic differentiation of rBMSCs, indicating that they are effective to treat the patients with delayed fracture healing.