富勒醇拮抗地塞米松诱导的骨髓间质干细胞氧化应激和成脂分化

Fullerol antagonizes deamethasone-induced oxidative stress and adipogenesis of bone marrow mesenchymal stem cells and enhances osteogenesis

目的 观察地塞米松（DEX）对骨髓间质干细胞（D1细胞系）成脂分化和成骨分化的影响,并探讨抗氧化剂富勒醇的干预作用.方法 将D1细胞随机分为：对照组、DEX组、DEX±谷胱甘肽组、DEX±富勒醇组,分组孵育细胞14d后,采用流式细胞仪检测细胞内活性氧簇（ROS）的含量；孵育21d后用油红O染色检测细胞的成脂分化；实时定量聚合酶链反应（Real-time PCR）检测aP2、过氧化物酶体增殖物激活受体γ（PPARγ）、核心结合蛋白因子-2（Runx2）、骨钙素、超氧化物歧化酶（SOD）、过氧化氢酶基因的表达.结果 第14天,同对照组比较,经地塞米松处理的细胞中,染色阳性细胞的百分率由91.0％增加到94.7％,染色阳性细胞最低的为经1.0μmmol/L富勒醇处理组.第21天,经DEX处理组的吸光度值为0.439±0.015,较对照组（0.169 ±0.020）及同时应用抗氧剂组均高.最低的为经1.0 μmmol/L富勒醇处理组.DEX明显增加D1细胞中aP2和PPARγ基因的表达,降低Runx2、骨钙素、SOD和过氧化氢酶基因的表达,增加ROS水平,促进成脂分化,富勒醇可明显拮抗DEX诱导的上述效应.结论 富勒醇可能通过降低氧化应激水平,进而减少成脂分化、增加成骨分化.

Objective To evaluate the effect of fullerol,a powerful antioxidant,on adipogenic and osteogenic differentiation of a mouse bone marrow-derived multipotent cell line,D1.Methods D1 cells were divided into five groups：D1 cells; D1 cells treated with 1 x 10-5 mol/L dexamethasone （DEX） ; （3） D1 cells treated with 1 x 10-5 mol/L DEX and 1 x 10-5 mol/L glutathione （GSH） ; （4） D1 cells treated with1 ＆#215;10-5 mol/L DEX and 1 ＆#215;10-7 mol/L fullerol; （5） Dl cells treated with 1 ＆#215;10-7 mol/L DEX and 1 x10-6 mol/L fullerol.Reactive oxygen species （ROS） were measured by staining the cells at day 21 with a fluorescent probe,2＆#39;,7＆#39;-dichlorodihydrofluorescin diacetate （DCFH-DA）.Lipid droplets within cells were assessed by staining with Oil Red O at day 21.The gene expression was detected by using quantitative realtime quantitative polymerase chain reaction （Real-time PC R）.Results The 14 d,compared with the control group （91.0%）,the percentage of positive cells was 94.7% in dexamethasone-treated cells,the lowest positive cells was treated by 1.0 μmmol/L fullerol.The 21 d,the highest optical density value of DEX-treated group was 0.439 ±0.015,compared with the control group （0.169 ±0.020） and the other which treated with antioxidant.The lowest was treated by 1.0 μmmol/L fullerol.DEX increased the expression of aP2 andperoxisome proliferator activated receptor γ （PPARγ） gene significantly,reduced the expression of related transcription factor-2 （Runx2）,osteocalcin,superoxide dismutase （SOD） and catalase gene,increased the levels of ROS,and promoted adipogenic differentiation,which could be antagonized significantly by fullerenol.Conclusion Fullerol antagonizes dexamethasone-induced oxidative stress and adipogenesis by reducing the leveal of ROS.