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糖尿病环境下成骨细胞KDM6B表达的变化

Study on the Expression of KDM6B of Osteoblast in Diabetic Environment

    摘要
  • 摘要:
      目的  研究糖尿病环境下骨髓间充质干细胞(BMSCs)的成骨分化能力及组蛋白去甲基化酶KDM6B表达的变化情况。
      方法  制备糖尿病大鼠模型，分离培养糖尿病模型大鼠及正常大鼠来源的BMSCs，BMSCs以高糖、晚期糖基化终末产物(advanced glycosylation products，AGE)模拟糖尿病环境，即将BMSCs分为6组：糖尿病组(糖尿病大鼠来源)和正常组(正常对照大鼠来源)，高糖组(含D-葡萄糖30 mmol/L)和正常组(含普通浓度的D-葡萄糖5.5 mmol/L作为高糖对照)，AGE组(含AGE 300 μg/mL)和牛血清白蛋白(BSA，作为AGE对照)组(含BSA 300 μg/mL)。除糖尿病组BMSCs为糖尿病大鼠来源，其余BMSCs为正常对照大鼠来源。各组BMSCs分别成骨诱导7 d后，进行碱性磷酸酶(alkaline phosphatase，ALP)染色，观察BMSCs的诱导骨向分化能力；以实时荧光定量PCR(qRT-PCR)检测各组BMSCs的成骨相关基因Runt相关转录因子2(Runt-related transcription factor 2，Runx2)、组蛋白去甲基化酶KDM6B mRNA的表达，以Western blot检测H3K27Me3蛋白的表达。
      结果  糖尿病组、高糖组及AGE组BMSCs的ALP染色细胞数均少于其相应对照组(P < 0.05)，Runx2、KDM6B mRNA表达也均低于其对照组(P < 0.05)，而H3K27Me3蛋白表达均高于其对照组(P < 0.05)。
      结论  糖尿病环境下BMSCs成骨分化能力减弱，且Runx2 mRNA表达受到抑制，可能与KDM6B的表达抑制后H3K27Me3表达增加有关。

     

    Abstract:
      Objective  To investigate the ability of osteogenic differentiation and the expression of histone demethylases KDM6B in bone marrow mesenchymal stem cells (BMSCs) in diabetic environment.
      Methods  Diabetic model rats was successfully established, and BMSCs from diabetic model rats and normal rats were isolated and cultured for further study. When cultured cells, we added high concentration of glucose and advanced glycosylation products (AGE) in the medium to imitating the diabetic environment. BMSCs were divided into 6 groups: diabetes group (derived from diabets SD rats), normal group (derived from normal SD rats), high glucose group (30 mmol/L D-glucose), normal glucose group (5.5 mmol/L D-glucose), AGE group (AGE 300 μg/mL) and BSA group (BSA 300 μg/mL). BMSCs in diabetes group were derived from diabetes SD rats, while others were derived from normal SD rats. After 7 d of osteogenic induction, the cells were examined the ability of osteogenic differentiation by alkaline phosphatase (ALP) staining, the transcription levels of Runt-related transcription factor 2 (Runx2) and KDM6B were determined by RT-PCR, and the expression levels of H3K27Me3 protein were examined by Western bolt.
      Results  Compared with the control groups, the numbers of ALP stained cells and the mRNA levels of Runx2 and KDM6B in diabetes group, high glucose group and AGE group were all decreased (P < 0.05), while H3K27Me3 protein expression levels were all increased (P < 0.05).
      Conclusion  The ability of osteogenic differentiation of BMSCs in diabetic environment was weakened, and the expression of Runx2 mRNA was inhibited, which may be related to the increased expression of H3K27Me3 after the inhibition of KDM6B expression.

     

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