利福平抑制地塞米松诱导骨髓间充质干细胞成脂分化的机制研究

目的 明确利福平对糖皮质激素诱导的大鼠骨髓间充质干细胞(bone marrow stroma cell, BMSC)成脂分化的作用。方法 体外培养Sprague-Dawley大鼠BMSC并随机分为4组: 5μg/ml利福平+10-6mol/L地塞米松(A),PBS+10-6mol/L地塞米松(B),5μg/ml利福平(C)和PBS(D)。分别应用罗丹明123结合流式细胞仪方法和酶联免疫吸附法检测5μg/ml利福平对BMSC的P糖蛋白活性和胞内地塞米松蓄积浓度影响。各组孵育14d后,分别进行油红O染色和碱性磷酸酶染色,定量检测三酰甘油含量、碱性磷酸酶活力、过氧化物酶体增殖物激活受体γ(PPARγ) mRNA和Runt相关转录因子2(Runx2)mRNA表达。结果 利福平减少BMSC胞内罗丹明123强度和胞内地塞米松蓄积量(P＜0.01)。B组诱导BMSC成脂分化、PPARγ mRNA表达增加和三酰甘油含量升高。A组、C组和D组BMSC未发生成脂分化,PPARγ mRNA表达增加和三酰甘油含量无统计学差异。A组BMSC碱性磷酸酶活性和Runx2 mRNA表达最强(P＜0.01),B组碱性磷酸酶活性和Runx2 mRNA表达下降(P＜0.01)。结论 利福平上调BMSC的P糖蛋白活性,抑制糖皮质激素诱导的成脂分化。
Objective To investigate the effect of rifampicin on dexamethasone-induced adipogenesis of bone marrow stem cells (BNSCs) and its mechanism. Methods BMSCs were isolated from Sprague-Dawley rats and randomly divided into 4 groups. Group A was treated with 5μg/ml rifampicin and 10-6mol/L dexamethasone; group B was treated with PBS and 10-6mol/L dexamethasone; group C and group D were treated with 5μg/ml rifampicin and PBS, respectively. P-glycoprotein activity and intracellular dexamethasone accumulation with the pretreatment of 5μg/ml rifampicin were determined by flow cytometry and enzyme linked immunosorbent assay (ELISA),respectively. After 14 days, BMSCs in all groups were observed under light microscope after Oil red O and alkaline phosphatase staining; triglyceride and alkaline phosphatase (AKP) activity levels were measured, peroxisome proliferator activated receptor γ (PPARγ) mRNA and runt-related transcription factor 2 (Runx2) mRNA level were detected by RT-fqPCR. Results Intracellular rhodamine123 fluorescence and dexamethasone level significantly decreased after 5μg/ml rifampicin treatment. Adipogenesis was showed in group B with increased PPARγ mRNA and triglyceride level. There were not adipogensis in group A, group C and group D. AKP staining and levels in group A were higher than those in group B. Conclusion Rifampicin can enhance P-gp activity and inhibit glucocorticoid-induced adipogenesis in BMSCs