高糖环境下GATA4通过调节RUNX2影响成骨细胞增殖分化的研究

GATA4 affects the proliferation and differentiation of osteoblasts by regulating Runx2 under high glucose condition

目的:探讨高糖环境下GATA锌指转录因子蛋白4(GATA4)对成骨细胞增殖分化的影响,及对转录因子蛋白2(Runx2)的调控作用。方法:取MC3T3-E1成骨细胞,在高糖(25 mmoL/L)的条件下分别培养1 d、4 d、7 d、14 d,免疫印迹法(Western blotting)法检测GATA4、Runx2蛋白表达水平。将MC3T3-E1成骨细胞分为空白对照组、高糖诱导组、GATA4过表达腺病毒(Ad-GATA4)组、GATA4空载腺病毒组(Ad-eGFP)组,CCK-8法检测各组细胞的存活率,碱性磷酸酶(ALP)染色法及茜素红染色法检测细胞分化能力;Western blot法检测各组细胞GATA4、Runx2、成骨细胞特异性转录因子(OSX)、骨唾液蛋白(BSP)蛋白表达水平。共转染Ad-GATA4及Runx2干扰腺病毒(Ad-si-Runx2),将MC3T3-E1成骨细胞分为Ad-GATA4-si-Runx2组、AdGATA4-eGFP2组、Ad-eGFP-si-Runx2组、Ad-eGFP-eGFP2组及未转染组(高糖组),高糖条件下培养7 d后检测GATA4、Runx2、OSX、BSP蛋白表达水平。结果:随着高糖培养时间延长,细胞中GATA4、Runx2蛋白表达持续降低,在第7天降低至最低,选取高糖培养7 d为后续培养条件。转染Ad-GATA4后,与空白对照组比较,高糖诱导组细胞矿化结节形成较少,细胞存活率、ALP活性、GATA4、Runx2蛋白表达、OSX、BSP蛋白表达降低(P<0.05);与高糖组相比,Ad-GATA4组细胞矿化结节形成较多,细胞存活率、ALP活性、GATA4、Runx2蛋白表达、OSX、BSP蛋白表达升高(P<0.05),Ad-eGFP组与高糖诱导组相比上述指标差异无统计学意义(P>0.05)。Ad-GATA4与Ad-si-Runx2共转染后,与高糖诱导组相比,Ad-GATA4-eGFP2组细胞GATA4、Runx2、OSX及BSP蛋白表达升高(P<0.05);Ad-eGFP-si-Runx2细胞Runx2、OSX及BSP蛋白表达降低(P<0.05),GATA4蛋白表达变化差异无统计学意义(P>0.05)。与Ad-GATA4-eGFP2组比较,Ad-GATA4-si-Runx2细胞Runx2、OSX及BSP蛋白表达降低(P<0.05),GATA4蛋白表达变化差异无统计学意义(P>0.05)。结论:高糖可抑制成骨细胞增殖分化,伴随GATA4、Runx2蛋白表达降低。过表达GATA4则可促进成骨细胞在高糖环境中的增殖、分化,其可能与激活Runx2表达有关。

Objective: To investigate the effects of GATA zinc finger transcription factor protein 4(GATA4) on the proliferation and differentiation of osteoblasts under high glucose condition and its regulation on transcription factor protein 2(Runx2). Methods: MC3 T3-E1 osteoblasts were cultured under high glucose(25 mmol/L) conditions for 1, 4, 7, and 14 days respectively. MC3 T3-E1 osteoblasts were divided into blank control group, high-glucose induction group, GATA4 over expression adenovirus(Ad-GATA4) group and GATA4 empty adenovirus(AdeGFP) group. The cell survival rate was detected by CCK-8 method. The cell differentiation ability was detected by alkaline phosphatase(ALP) staining and alizarin red staining. The expression levels of GATA4, Runx2,osteoblast specific transcription factor(OSX) and bone sialoprotein(BSP) proteins were detected by Western blotting. After co-transfection with Ad-GATA4 and Runx2-interfered adenovirus(Ad-si-Runx2), the MC3 T3-E1 osteoblasts were divided into Ad-GATA4-si-Runx2 group, Ad-GATA4-eGFP2 group, Ad-eGFP-si-Runx2 group, Ad-eGFP-eGFP2 group and non transfection group(high glucose group), and the expression levels of GATA4, Runx2, OSX and BSP proteins were detected after 7 days of high glucose induction. Results: With the high-glucose induction time prolonged, the expression of GATA4 and Runx2 proteins decreased continuously, then decreased to the minimum on the 7 th day. Therefore, highglucose culture for 7 days was selected as subsequent culture condition. After Ad-GATA4 transfection, there were fewer mineralized nodules in the high glucose group, and the cell survival rate, ALP activity and the expression of GATA4, Runx2, OSX and BSP proteins in the high-glucose induction group were lower than those in the blank control group(P<0.05). The mineralized nodules were morein the Ad-GATA4 group compared with high glucose group, and the cell survival rate, ALP activity and the expression of GATA4, Runx2, OSX and BSP proteins in the Ad-GATA4 group were higher(P<0.05). There was no significant difference in the above indexes between Ad-eGFP group and high-glucose induction group(P>0.05). After co-transfection of Ad-GATA4 and Ad-siRunx2, the expression of GATA4, Runx2, OSX and BSP proteins in the Ad-GATA4-eGFP2 group was significantly higher than that in high-glucose induction group(P<0.05), while the expression of Runx2, OSX and BSP proteins in Ad-eGFP-si-Runx2 cells was lower(P<0.05), but the difference in GATA4 protein expression was not statistically significant(P>0.05). The expression of Runx2, OSX and BSP proteins in Ad-GATA4-si-Runx2 cells was lower than that in Ad-GATA4-eGFP2 group(P<0.05), but the difference in GATA4 protein expression was not statistically significant(P>0.05). Conclusion: High glucose condition can inhibit the proliferation and differentiation of osteoblasts, and down-regulate GATA4 and Runx2 expression. Over expression of GATA4 can promote the proliferation and differentiation of osteoblasts under high glucose condition, which may be related to the activation of Runx2 expression.