直流电场对炎症微环境下的rBMSCs成骨的影响

Effects of direct current electric field on osteogenic differentiation of rBMSCs under inflammatory microenvironment

目的:探究直流微电场(DCEF)对炎症微环境下的大鼠骨髓间充质干细胞(rBMSCs)成骨分化作用的影响。方法:提取、培养及鉴定rBMSCs;以10mg/ml的TNF-α诱发细胞炎症反应,采用ELISA及实时定量PCR检测IL-1β及TNF-α的分泌量及基因表达;对正常rBMSCs(H-rBMSCs)组及炎症微环境下rBMSCs(H+cy tok ines)组加载DCEF,MTT法测生长曲线,茜素红染色及钙离子沉积量检测,实时定量PCR检测成骨基因BSP、OCN、Runx2的表达,Western blot检测成骨蛋白ALP及Runx2的表达量。结果:H+cytokines组细胞IL-1β及TNF-α的分泌量均明显高于H-rBMSCs组(35.6±3.0比12.2±1.5;49.2±3.5比14.9±2.1,P<0.05);H+cy tok ines组IL-1β及TNF-α基因表达水平分别是H-rBMSCs组2.5倍、2.2倍;H+cy tok ines+DCEF组及H-rBMSCs+DCEF组BSP、OCN、Runx2基因表达水平及ALP、Runx2蛋白表达水平分别高于H+cytokines组及H-rBMSCs(P<0.05),H+cytokines+DCEF组部分成骨能力指标高于H-rBMSCs(ALP蛋白表达水平:0.95±0.08比0.43±0.04,R unx 2蛋白表达水平:0.85±0.08比0.31±0.04;P<0.05)。结论:DCEF可促进炎症微环境下的rBMSCs增殖及成骨分化。

Objective：To investigate the effect of direct current electric field（DECF） on the osteogenic differentiation of rat bone marrow mesenchymal stem cells（rBMSCs） under inflammatory microenvironment. Methods：Rat tibia BMSCs were extracted, cultured and identified. The cell inflammatory response was induced by 10mg/ml TNF-α. Then, the secretions and the gene expression levels of IL-1β and TNF-α were measured by ELISA and real-time quantitative PCR. DCEF was loaded on healthy rBMSCs（H-rBMSCs） and rBMSCs under inflammatory microenvironment（H ＋ cytokines）, then the growth curves were measured by MTT method, the deposition amounts of calciumions were measured after alizarin red stained, the expressions of key osteogenic genes（BSP, OCN and Runx2） were detected by real-time quantitative PCR, the expressions of key osteogenic proteins（ALP and Runx2） were detected by Western blot. Results：The secretions of IL-1βand TNF-α in H＋cytokines group were significantly higher than those in H-rBMSCs group.（35.6±3.0 VS 12.2±1.5, 49.2±3.5VS14.9±2.1, P〈0.05） The expression levels of the two genes were 2.5 fold and 2.2 fold.（P〈0.05） The BSP, OCN, Runx2 gene expression levels and ALP, Runx2 protein expression levels of H＋cytokines＋DCEF group and H-rBMSCs＋DCEF group were significantly higher than those of H＋cytokines group and H-rBMSCs group, respectively.（P〈0.05） Some of the osteogenic abilities of H＋cytokines＋DCEF group were stronger than those of H-rBMSCs.（ALP protein expression levels：0.95±0.08 vs 0.43±0.04, Runx2 protein expression levels：0.85±0.08 vs 0.31±0.04, P〈0.05） Conclusion：DCEF can promote proliferation and osteogenic differentiation of rBMSCs in inflammatory microenvironment.