异补骨脂素对小鼠骨髓基质干细胞成骨和成脂分化影响及其机制的相关研究

Effects of isopsoralen on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells in mice

目的探索异补骨脂素对体外培养小鼠骨髓基质干细胞（BMSCs）成骨和成脂分化的影响及其作用机制。方法选取18只2个月大的无特定病原体级雌性C57／BL6小鼠，分离BMSCs。通过细胞培养、细胞增殖、碱性磷酸酶（ALP）染色实验、油红O染色实验建立并鉴定成骨、成脂细胞诱导，诱导14d后检测核心结合蛋白因子2（RUNX2）、骨钙素（OCN）、过氧化物酶体增殖物激活受体1（PPAR-γ）、CCAAT增强子结合蛋白β（C／EBP-β）确定浓度分别为0（对照组）、5、10、20μmol／L异补骨脂素的作用；并通过蛋白免疫印迹实验检测BMSCs成骨诱导及成脂诱导信号通路中P-S6（S235／236）、4E／BP1（Thr37／46）的表达。结果成骨诱导14d后10、20μmol／L组的BMSCs内ALP阳性表达量分别较对照组、5μmol／L组有增加，20μmol／L组ALP表达最强。5、10、20μmol／L组RUNX2、OCN的表达均较对照组增加，差异有统计学意义（P〈0．05），20μmol／L组RUNX2、OCN表达量最大。成脂诱导14d后5、10、20μmol／L的异补骨脂素浓度均能抑制BMSCs中PPAR-γ、C／EBP-β的表达，差异有统计学意义（P〈0．05），而20μmol／L的异补骨脂素抑制效果最强。成骨诱导14d后P—S6（S235／236）明显下降，且20μmol／L组最低，而4E／BP1（Thr37／46）的表达明显上升，对照组表达是最低。成脂诱导14d后P-S6（S235／236）表达明显上升，对照组P-S6（S235／236）表达最低，而4E／BP1（Thr37／46）的表达随异补骨脂素浓度的升高明显下降，且20p,mol／L组最低。结论异补骨脂素增强小鼠BMSCs成骨作用，抑制其向脂肪细胞分化，并证实异补骨脂素是通过抑制mTORC1信号通路活性抑制BMSCs向脂肪细胞分化。

Objective To explore the effects of isopsoralen on the osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells （BMSCs） in mice and their mechanisms. Methods BMSCs were isolated from 18 2-month old female C57/BL6 mice that were specific pathogen free. Induced osteoblasts and fat cells were established and identified through cell culture, cell proliferation assay, alkaline phosphatase （ALP） staining and Oil Red O staining. After 14 days＇ induction, the core binding protein 2 （RUNX2）, osteocalcin （OCN）, a peroxisome proliferator activated receptor gamma （PPAR-γ） and CCAAT enhancer binding protein beta （C/EBP-β） were detected to determine the role of isopsoralen in groups of isopsoralen concentrations of 0, 5, 10 and 20 μmol/L. Western blotting was used to detect the expression of P-S6 （S235/236） and 4E/BP1 （Thr37/46） in the signal pathways for osteogenic induction and adipogenic induction of BMSCs. Results After osteogenic induction for 14 days： the positive expression of ALP in BMSCs in the 10 and 20 μmol/L groups were increased compared to the 0 and 5 μmol/L groups, with the highest in the 20 μmol/L group; the expression of RUNX2 and OCN in the 5, 10 and 20 μmol/L groups was significantly increased compared to the 0 μmol/L group, with the highest in the 20μmol/L group （P 〈 0. 05） . After adipogenic induction for 14 days： the isopsoralen in the 5, 10 and 20 μmol/L groups significantly inhibited the expression of PPAR-γ and C/EBP-B in BMSCs, with the most in the 20 μmol/L group （ P 〈0. 05） . After osteogenic induction for 14 days： the expression of P-S6 （S235/236） was obviously decreased, with the lowest in the 20 μmol/L group; the expression of 4E/BP1 （Thr37/46） was obviously increased, with the lowest in the 0μmol/L group. After adipogenie induction for 14 days： the expression of P-S6 （S235/236） was obviously increased, with the lowest in the 0 μmol/L group; the expression of 4E/ BP1（Thr37/46） was obviously decreased, with the lowest in the 20 μmol/L group. Conclusions Isopsoralen may enhance osteogenesis of BMSCs and inhibit BMSCs from differentiation into fat cells. The present experiment has also confirmed that isopsoialen inhibits the adipogenic differentiation of BMSCs by inhibiting the activity of mTORC 1 signaling pathway.