高葡萄糖培养增加H9C2成心肌细胞G蛋白偶联受体激酶2基因表达

High glucose up-regulates GRK2 gene expression in H9C2 cardiomyoblasts in vitro

目的前期临床研究发现2型糖尿病患者淋巴细胞G蛋白偶联受体激酶2(GRK2)基因表达升高,为进一步探讨GRK2表达升高的分子机制,检测高糖对GRK2基因表达的影响。方法H9C2成心肌细胞随机分成5组分别加入以下培养液:空白对照组和不同葡萄糖梯度浓度组(0,5.5,12.5,25,33mmol/L),培养72h后采用RT-PCR,WESTERN BLOTTING分别测定GRK2-mRNA和磷酸化Akt(Ser473)蛋白含量比值。结果随着葡萄糖浓度升高,H9C2成心肌细胞的GRK2-mRNA表达水平呈剂量依赖性增加,pearson相关分析提示提示两者间存在直线相关(R=0.683,P<0.001),采用Student Newman-Keuls'q检验,各葡萄糖培养组与无糖培养比较均存在统计学差异(P=0.028,P=0.009,P<0.001)。而磷酸化Akt(Ser473)蛋白表达水平减少,各组间比较存在统计学意义(F=369.1,P<0.001),回归分析和pearson相关分析提示葡萄糖浓度与磷酸化AKt(Ser473)蛋白的表达呈线性负相关(R=-0.913,P<0.001)。结论高糖培养使GRK2表达升高,可能是参与心肌细胞葡萄糖利用受损的重要机制之一。

Objective To study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro. Methods H9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose （with the osmotic pressure adjusted with monnitol）. Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt（Ser473） protein level was measured by Western blotting. Results Glucose in the culture medium （5.5 to 33 mmol/L） concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt （ser473） level in in H9C2 cardiomyoblasts. Conclusion Increased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.