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目的通过检测Akt在大鼠心肌细胞退出细胞周期过程中的表达及Akt表达载体转染后大鼠心肌细胞有丝分裂的改变,探讨Akt对心肌细胞增殖的影响。方法生后不同发育阶段和成年健康Wistar大鼠各5只,用RT-PCR和免疫印迹法,检测心肌组织Akt的mRNA和蛋白磷酸化水平改变;培养大鼠H9c2(2-1)心肌细胞,通过胰岛素刺激和真核表达载体pCIS2-Akt-WT转染两种方式激活细胞内Akt,采用免疫沉淀方法检测细胞内磷酸化组蛋白H3表达改变,观察Akt对心肌细胞有丝分裂的影响。结果大鼠生后7d到2周心肌组织Akt mRNA表达和蛋白磷酸化水平明显下降(P<0.01),激活的Akt促使大鼠H9c2(2-1)心肌细胞磷酸化组蛋白H3表达显著增加(P<0.01)。结论 Akt能促进可增殖心肌细胞的有丝分裂。
Objective To investigate the effect of Akt on cardiomyocyte proliferation by detecting the expression of Akt during cardiomyocyte exit cell cycle and mitochondrial alterations of rat cardiomyocytes after Akt expression vector transfection. Methods Five adult Wistar rats were born at different developmental stages after birth. The mRNA and protein phosphorylation levels of Akt in myocardium were detected by RT-PCR and Western blotting. The rat H9c2 (2-1) cardiomyocytes were cultured, Activation of intracellular Akt by insulin stimulation and eukaryotic expression vector pCIS2-Akt-WT was used to detect the expression of phosphorylated histone H3 by immunoprecipitation, and the effect of Akt on mitosis of cardiomyocytes was observed. Results Akt mRNA and protein phosphorylation in myocardium were significantly decreased at 7d and 2d after birth (P <0.01). Activated Akt significantly increased phosphorylated histone H3 expression in H9c2 (2-1) P <0.01). Conclusions Akt can promote the mitosis of proliferative cardiomyocytes.