地塞米松对骨髓间充质细胞增殖及成骨、成脂分化的效应

摘要

目的 探讨不同浓度地塞米松(dexamethasone,DEX)作用不同时间对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的增殖及成骨、成脂分化的影响.方法 体外分离培养大鼠BMSCs,将状态良好的第三代细胞接种于96孔板中,随机分组为对照组(不加入DEX)及不同浓度(1 nmol/L、10 nmol/L、0.1μmol/L、1μmol/L)DEX作用组,培养1、3、5、7、9 d后,分别用细胞增殖检测(CCK-8)法检测细胞增殖状况.将BMSCs接种于6孔板中,随机分为对照组及不同浓度(1 nmol/L、10 nmol/L、0.1μmol/L、1μmol/L)DEX作用组,培养7、14 d后分别用荧光定量PCR方法检测成骨、成脂相关基因的表达,培养14 d后用Western blot检测成骨、成脂相关蛋白的表达.培养5 d后进行碱性磷酸酶(alkaline phosphatase,ALP)染色,培养14 d后进行油红"O"染色、茜素红染色.结果 较低浓度的DEX促进BMSCs增殖,而较高浓度DEX抑制BMSCs增殖.干预早期,较低浓度的DEX促进BMSCs成骨分化而高浓度DEX抑制其成骨分化;干预晚期,各浓度DEX均促进BMSCs成骨指标的表达,但是不能诱导BMSCs钙质沉积.DEX浓度依赖性地促进BMSCs成脂相关指标基因和蛋白的表达,并诱导BMSCs细胞内脂质沉积.结论 DEX可直接影响BMSCs增殖及向成骨、成脂方向分化,这种效应存在浓度和干预时间依赖性.%Objective To study the effects of dexamethasone (DEX) on bone marrow mesenchymal stem cells (BMSCs) proliferation, osteogenic or adipogenic differentiation. Methods BMSCs were isolated and cultured from 4-weeks-old Sprague-Dawley rats. BMSCs of passage 3 were seeded into 96-well plates, and randomly divided into control group and DEX treated groups with different concentrations (1 nmol/L, 10 nmol/L, 0.1μmol/L, 1μmol/L). The proliferation was detected with CCK-8 kit at day 1, day 3, day 5, day 7, and day 9. The cells were seeded into 6-well plates, and treated with DEX of different concentrations (1 nmol/L, 10 nmol/L, 0.1 μmol/L, 1 μmol/L) or growth medium (control group) randomly. After culture for 7 days or 14 days, the osteogenic and adipogenic related genes were detected by real-time PCR. The protein expression was detected by Western blot. ALP histochemical staining was performed on the day 5. Alizarin Red S staining and Oil Red O staining were performed on the day 14. Results DEX of lower concentrations promoted the proliferation of BMSCs, while higher concentrations of DEX inhibited it. At early stage, DEX of lower concentrations promoted BMSCs osteogenic related genes, while higher concentrations of DEX inhibited them. At late stage, DEX of each concentration promoted the expression of osteogenic differentiation index. However, the DEX alone did not increase the number of calcium nodules of BMSCs. The adipogenic differentiation of BMSCs was promoted by DEX in a concentration dependent manner. Conclusion The proliferation and osteogenic or adipogenic differentiation of BMSCs can be affected by DEX time-and concentration-dependently.