论文部分内容阅读
目的 探讨抗氧化剂谷胱甘肽(glutathione,GSH)对激素诱导的人BMSCs成脂、成骨关键因子表达水平的影响.方法 取3例股骨颈骨折患者股骨近端骨髓,采用密度梯度离心联合贴壁法进行BMSCs的分离、培养和纯化.取第3代BMSCs分为5组:A组,单纯BMSCs组(1×105个/mL);B组,BMSCs(1×105个/mL)+10 μmol/L地塞米松;C组,BMSCs(1×105个/mL)+10 μmol/L地塞米松+5 μmol/L GSH;D组,BMSCs(1×105个/mL)+10 μmol/L地塞米松+10 μmol/L GSH;E组,BMSCs(1×105个/mL)+10 μmol/L地塞米松+50 μmol/L GSH.培养7d,采用流式细胞仪检测细胞内活性氧水平,RT-PCR检测细胞内超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(Catalase) mRNA表达水平,实时荧光定量PCR检测成脂转录因子过氧化物酶体增殖激活受体γ(peroxisome proliferator-activated receptors γ,PPAR-γ)、CCAAT增强结合蛋白(CCAAT/enhancer-binding family of proteins,C/EBP)和成骨转录因子Runx2、ALP基因表达情况;培养21d,采用油红O染色观察细胞成脂分化情况,Western blot检测相关蛋白表达水平.结果 B组细胞内活性氧水平显著高于其余各组,C组高于A、D、E组,D、E组高于A组(P＜0.05);D、E组间比较差异无统计学意义(P＞0.05).B组油红O染色阳性细胞明显多于其余各组,C、D、E、A组依次减少,各组吸光度(A)值比较差异均有统计学意义(P＜0.05).RT-PCR检测示,B组SOD和CatalasemRNA相对表达量较其余各组显著降低,C组低于A、D、E组(P＜0.05);A、D、E组间比较差异无统计学意义(P＞0.05).实时荧光定量PCR检测示,B组PPAR-γ、C/EBPmRNA相对表达量显著高于其余各组(P＜0.05);C组高于A、D、E组,D、E组高于A组,差异均有统计学意义(P＜0.05);D、E组间比较差异无统计学意义(P＞0.05).B组Runx2、ALP mRNA相对表达量显著低于其余各组,C组低于A、D、E组,D、E组低于A组,差异均有统计学意义(P＜0.05);D、E组间比较差异无统计学意义(P＞0.05).Western blot检测示,B组PPAR-γ、C/EBP蛋白相对表达量较其余各组显著提高,C、D、E、A组呈逐渐下降趋势,组间比较差异均有统计学意义(P＜0.05);B组Runx2、ALP蛋白相对表达量较其余各组显著下降,C、D、E、A组呈逐渐上升趋势,组间比较差异均有统计学意义(P＜0.05).结论 GSH可通过降低细胞内活性氧水平抑制人BMSCs成脂分化,增强成骨分化;在一定范围内,GSH浓度越高,效果越明显.“,”Objective To investigate the protective effect of the antioxidant glutathione (GSH) on the steroidinduced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs).Methods The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated,cultured,and purificated by density gradient centrifugation and adherent wall method in vitro.The third generation BMSCs were divided into 5 groups:group A,BMSCs (1 × 105 cells/mL);group B,BMSCs (1 x 105 cells/mL)+10 μmol/L dexamethasone;group C,BMSCs (1 × 105 cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH;group D,BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH;group E,BMSCs (1×105 cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH.After cultured for 7 days,the reactive oxygen species expression was detected by flow cytometry;the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR;the peroxisome proliferator-activated receptors γ (PPAR-γ),CCAAT/enhancer-binding family of proteins (C/EBP),Runx2,and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR.After cultured for 21 days,Oil red O staining was used to observe the adipogenesis differentiation of cells,and the expressions of related proteins were detected by Western blot.Results The reactive oxygen species expression in group B was obviously higher than in the other groups,in group C than in groups A,D,and E,and in groups D,E than in group A,all showing significant differences between groups (P＜0.05);but there was no significant difference between groups D and E (P＞0.05).The oil red O staining positive cells in group B were obviously more than the other groups,and groups C,D,E,and A decreased sequentially,the absorbance (A) values had significant differences between groups (P＜0.05).RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups,while in group C than in groups A,D,and E (P＜0.05),but there was no significant difference among groups A,D,and E (P＞0.05).Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups,while in group C than in groups A,D,and E,and in groups D,E than in group A (P＜0.05);but there was no significant difference between groups D and E (P＞0.05).The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups,while in group C than in groups A,D,and E,and in groups D,E than in group A (P＜0.05);but there was no significant difference between groups D and E (P＞0.05).Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups,and groups C,D,E,and A decreased sequentially,all showing significant differences between groups (P＜0.05).The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups,and groups C,D,E,and A increased sequentially,all showing significant differences between groups (P＜0.05).Conclusions GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level;and in a certain range,the higher the concentration of GSH,the more obvious the effect is.