左、右归丸及其拆方对骨髓间充质干细胞成骨分化的影响

摘要

目的:研究左、右归丸及其拆方含药血清对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化的作用.方法:分别以左归丸、右归丸、两方共同药、滋肾阴药、补肾阳药、阳性对照药补佳乐和蒸馏水制备的大鼠含药血清加诱导剂(地塞米松、维生素C和β-甘油磷酸钠)干预BMSCs,并以不含诱导剂的空白含药血清组为对照,共8组,采用改良钙钴法检测碱性磷酸酶(alkaline phosphatase,ALP)活性,茜素红法观察矿化结节的形成,并用real-time RT-PCR法检测核心结合因子α1(core binding factor alpha 1,Cbfα1)和Ⅰ型胶原(type Ⅰ collagen,Col Ⅰ)mRNA表达,用Western blotting法检测Cbfα1和Col Ⅰ蛋白表达.结果:左、右归丸及其拆方与阳性对照药补佳乐均可协同诱导剂诱导BMSCs成骨分化,其中,左归丸和滋肾阴药可显著促进ALP生成和矿化结节形成,上调Cbfα1和Col Ⅰ mRNA及蛋白表达(P＜0.05).结论:左、右归丸及其拆方含药血清能协同诱导剂促进BMSCs成骨分化,左归丸和滋肾阴药促进BMSCs成骨分化的作用优于右归丸和补肾阳药.%AIM:To observe the effects of the sera containing Zuogui pill,Yougui pill and their disassembled prescriptions on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).METHODS:Seven groups of sera containing Zuogui pill,Yougui pill,mutual drugs of Zuogui pill and Yougui pill,kidney-Yin-nourishing drugs,kidney-Yang-tonifying drugs,positive control drug estradiol valerate and distilled water,all of which were added inducer (dexamethasone,vitamin C and sodium β-glycerophosphate),and the other group of the control serum without inducer,were prepared,and were used to treat the BMSCs.The alkaline phosphatase (ALP) activity was detected by modified calciumcobalt staining.The mineralized nodules were counted with alizarin red staining.The expression of core binding factor alplat 1 (Cbfαt1) and collagen type 1 (Col Ⅰ) at mRNA and protein levels was determined by RT-PCR and Western blotting.RESULTS:All the sera containing Zuogui pill,Yougui pill and their disassembled prescriptions,and the positive control drug helped the inducer to induce the osteogenic differentiation of BMSCs.Especially,Zuogui pill and Yin-nourishing drugs of Zuogui pill remarkably increased ALP activity and formation of mineralized nodules,and up-regulated Cbfα1 and Col Ⅰ at mRNA and protein levels.CONCLUSION:Sera containing Zuogui pill,Yougui pill and their disassembled prescriptions help the inducer to induce osteogenic differentiation of BMSCs.Zuogui pill and Yin-nourishing drugs of Zuogui pill are better,indicating that nourishing Yin is superior to tonifying Yang for prevention and treatment of osteoporosis,according to the principles of traditional Chinese medicine.