陇中损伤散含药血清对大鼠骨髓间充质干细胞增殖与成骨性分化的影响

Effects of Serum Containing Longzhong Sunshang San on the Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Rats

目的:研究不同浓度的陇中损伤散含药血清对大鼠骨髓间充质干细胞(BMSCs)增殖与成骨性分化的影响。方法:取三月龄wistar大鼠骨髓,分离BMSCs进行原代培养,在第一次换液后分别加入2%,5%,10%的陇中损伤散含药血清,通过倒置显微镜观察细胞形态变化,MTT法测量骨髓间充质干细胞增殖情况;偶氮偶合法进行碱性磷酸酶阳性克隆(CFU-FALP)组织化学染色,vonKossa染色法测定钙化结节数目,免疫细胞化学技术检测Ⅰ型胶原表达;于不同时间进行ALP活性及钙盐沉积量的测定。结果:陇中损伤散含药血清对BMSCs的影响呈剂量依赖性,其中以浓度为10%时作用最强。MTT结果显示陇中损伤散含药血清能明显促进BMSCs增殖;损伤散含药血清还能显著促进其成骨性分化。结论::陇中损伤散活性代谢产物能促进BMSCs的增殖和成骨性分化;陇中损伤散对骨髓间充质干细胞的这种作用可能是其促进骨折愈合、防治骨质疏松的细胞学机制之一。

Objective：To study the effects of serum containing Longzhong Sunshang San （SLSS） on proliferation and osteogenic differentiation of bone marrow mysenchymal stem cells （BMSCs） in rats. Methods：BMSCs were separated from bone marrow collected from three-month-old Wistar rats and then co-cultured with 2%,5% and 10% SLSS. Morphological changes were observed with inverted microscope. MTT assay was performed to measure the proliferation of BMSCs. CFU -FALP staining was used to measure the number of CFU-F stained positive for ALP. Von Kossa staining and immunohistochemistry （IHC） techniques were separately used to measure the number of calcification tubercle and the expression of type I collogen. ALP activity and the calcium deposition level were measured at different periods of time. Results：SLSS enhanced the proliferation and osteogenic differentiation of BMSCs in a dose-dependent manner,with strongest effect at 10%. Conclusion：Longzhong Sunshang San could stimulate the proliferation and osteogenic differentiation of BMSCs,which might be one of the mechanisms of Longzhong Sunshang San in promoting the concrescence of bone fracture,preventing and treating osteoporosis.