糖皮质激素对DKK1基因调控骨髓间充质干细胞增殖分化的影响

The impact of glucocorticoid on proliferation and differentiation of bone marrow mesenchymal stem cells by regulating DKK1

目的观察不同浓度糖皮质激素刺激下骨髓间充质干细胞(BMSCs)中DKK1基因表达的变化及其对BMSCs增殖分化能力的影响。方法分离培养人BMSCs,分为对照组[不含地塞米松(Dex)]、Dex8组(含10-8mol/L Dex)及Dex6组(含10-6mol/L Dex)。分别用含0、10-8、10-6mol/L Dex培养液刺激BMSCs,采用Real-time PCR检测DKK1基因的表达量,并通过克隆形成实验检测其对BMSCs增殖能力的影响。分别用含0、10-8、10-6mol/L Dex的成骨诱导液及成脂诱导液刺激BMSCs,于3d后提取RNA,采用Real-time PCR检测DKK1基因、成骨基因(Runx2、OCN)及成脂基因(C/EBP、PPARγ)的表达量,并于3周后通过茜素红染色及油红O染色检测其对成骨、成脂分化能力的影响。结果与对照组及Dex8组比较,Dex6组DKK1基因的表达最高,而对应的克隆形成能力最低。成骨诱导环境下,Dex8组Runx2、OCN的表达最高,而DKK1的表达最低,茜素红染色只有Dex8组出现钙化结节。成脂诱导环境下,Dex6组C/EBP、PPARγ及DKK1的表达最高,油红O染色Dex6组出现大量的脂滴。结论高浓度糖皮质激素刺激下,DKK1表达上调,BMSCs增殖受抑。DKK1对BMSCs的骨向分化具有抑制作用,而对其脂向分化具有促进作用。糖皮质激素导致骨质疏松可能与其调控DKK1的表达、影响BMSCs的增殖和分化有关。

Objective To observe the influence of different concentrations of glucocorticoid on the expression of DKK1 gene and the effect of DKK1 on the proliferation and differentiation of bone marrow mesenchymal stem cells（BMSCs）.Methods The experiment consisted three groups,namely,control group（no Dex）,Dex8 group（10-8mol/L Dex）,and Dex6 group（10-6mol/L Dex）.Culture fluids with different concentrations of Dex,including 0,10-8,and 10-6mol/L,were used to stimulate BMSCs.Real-time PCR was used to detect the expression of the DKK1 gene,and clone formation experiment was used to detect the influence on the proliferation of BMSCs.Osteogenic-and adipogenic-induced media were used which contained 0,10-8,and 10-6mol/L Dex,respectively,to stimulate BMSCs,and to extract RNA after 3d.Real-time PCR was used to detect the expression of DKK1 genes,osteogenic genes（Runx2 and OCN）,and adipogenic genes（C/EBP and PPARγ）.After three weeks,Alizarin red staining and Oil red O staining were adopted to detect the influence on osteogenic and adipogenic differentiations.Results The expression of DKK1 was the highest,whereas the corresponding cloning formation ability was the lowest,after the stimulation of glucocorticoid with high concentration.The expressions of Runx2 and OCN under osteogenic induction in Dex8 group were the highest,whereas the expression of DKK1 was the lowest.Based on Alizarin red staining,calcified cortical tubers were only found in Dex8 group.The expressions of C/EBP and PPARγ under adipogenic induction in Dex6 group were the highest,so was the expression of DKK1.With Oil red O staining,there were abundant lipid drops in Dex6 group.Conclusion The expression of DKK1 was upregulated under high concentration of glucocorticoid,but it inhibited the proliferation of BMSCs.DKK1 inhibited osteogenic differentiation but promoted adipogenic differentiation of BMSCs.Osteoporosis after glucocorticoid administration may be ascribed to the fact that glucocorticoid regulates the expression of DKK1 and affects the proliferation and differentiation of BMSCs.