Zeste基因增强子同源物2抑制剂GSK126促进骨髓间充质干细胞成软骨分化

Effect of GSK126 on the chondrogenic differentiation of mesenchymal stem cells

目的观察Zeste基因增强子同源物2(EZH2)选择性抑制剂GSK126对骨髓间充质干细胞(BMSCs)成软骨分化的影响。方法体外分离培养BMSCs,取第3代细胞进行实验。噻唑蓝(MTT)检测确定GSK126的最适浓度。将细胞消化、悬浮后,以5×106/ml的细胞密度进行高密度培养,用含有或不含GSK126的软骨诱导液诱导培养3周。免疫组织化学、甲苯胺蓝染色检测Ⅱ型胶原、蛋白多糖的表达。实时定量聚合酶链反应(Real-time PCR)检测Ⅱ型胶原、蛋白多糖和性别决定区Y框蛋白9(SOX9) mRNA的表达。Western blot检测H3K27me3的蛋白含量。结果5μmol/L GSK126无明显细胞毒作用,被选为本实验最适浓度。组织学染色显示实验组微团中的细胞体积较对照组稍大,细胞密度稍低。两组微团均有Ⅱ型胶原、蛋白多糖的合成与分泌,且实验组的表达最为强烈,实验组Ⅱ型胶原吸光度(A)值为0.368 5±0.040 5,对照组A值为0.132 2±0.021 1。Real-time PCR检测显示,GSK126干预3周后实验组Ⅱ型胶原、蛋白多糖和SOX9基因的表达量明显高于对照组(P<0.01)。Western blot检测显示对照组H3K27me3蛋白水平显著高于实验组,GSK126的干预使得H3K27me3含量明显降低,实验组A值为0.326 5±0.030 2,对照组A值为0.674 2±0.056 2。结论 GSK126通过降低细胞内H3K27me3蛋白水平,有效地促进BMSCs成软骨方向分化。

Objective To investigate the effect of enhancer of Zeste homolog (EZH2) inhibitor GSK126 on the chondrogenic differentiation of the bone marrow-derived mesenchymal stem cells (BMSCs).Methods BMSCs were isolated by ficoll density-gradient centrifugation. The cells at passage 3 were used in this study. The optimal concentration of GSK126 was confirmed by methyl thiazol tetrazolium (MTT). On reaching confluence, cells were digested, suspended and then centrifuged at a density of 5×106/ml to form pellets. The pellets were cultured for 3 weeks with CM supplemented with GSK126 or not. The expression of type Ⅱ collagen and glycosaminoglycan (GAG) was detected by immunohistochemistry and toluidine blue staining, respectively. Real-time quantitative polymerase chain reaction (Real-time PCR) was performed to detect the content of type Ⅱ collagen, sex determining region Y box 9 (SOX9) and aggrecan mRNA, and the amount of H3K27me3 protein was assayed by Western blotting.Results 5 μmol/L GSK126 was regarded as the optimal concentration since the cytotoxic effect has not been observed. The cell volumn in the experiment group was larger than that in the control group, with lower cell density. The histological staining showed that type Ⅱ collagen and GAG were synthesized and secreted in all the pellets with stronger deposit in the experiment group. The absorbance (A) of experiment group and control group was 0.368 5±0.040 5 and 0.132 2±0.021 1 respectively. Real-time PCR showed that the mRNA expression of type Ⅱ collagen and aggrecan in the experiment group was significantly higher than that in the control group. High level of H3K27me3 protein was noted in the control group, with the A value 0.6742±0.056 2. Treatment with 10 μmol/L GSK126 obviously decreased the amount of H3K27me3, with the A value of 0.326 5±0.030 2.Conclusion GSK126 obviously promoted the chondrogenic differentiation of BMSCs by demethylation of H3K27me3.