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目的:建立大鼠心肌缺血再灌注损伤模型和氧化应激损伤细胞模型,研究抗肌萎缩蛋白Dp71在损伤心肌及细胞中的表达,探讨其拮抗心肌细胞凋亡的作用及其分子机制。方法:阻断SD大鼠冠状动脉左前降支血流30 min后恢复血流复制心肌缺血再灌注损伤模型,观察心肌形态学、LDH变化及心肌细胞凋亡;检测再灌注不同时间心脏Dp71蛋白和m RNA表达。建立H9c2细胞氧化应激损伤模型,检测H_2O_2刺激后细胞中Dp71蛋白和m RNA的表达。H9c2细胞转染Dp71过表达质粒,流式细胞术检测Dp71高表达对H_2O_2诱导H9c2细胞凋亡率的影响。Western blot检测过表达Dp71对细胞中lamin B1和Bcl-2蛋白表达的影响;对H_2O_2刺激下lamin B1和Bcl2蛋白表达改变的影响。结果:与假手术组相比,再灌注损伤组HE染色心肌出现明显形态改变,LDH明显增加,IR组心肌细胞凋亡数显著增加。IR后各时点Dp71 m RNA和蛋白表达水平均增加(P<0.05)。0.2 mmol/L H_2O_2刺激细胞16 h Dp71蛋白及m RNA表达明显升高(P<0.05)。H9c2细胞中转染Dp71过表达质粒可抑制H_2O_2所诱导的细胞凋亡;H9c2细胞中过表达Dp71引起lamin B1和Bcl2表达增高,高表达的Dp71可以抑制过氧化氢刺激引起的lamin B1和Bcl2表达下降。结论:Dp71 m RNA和蛋白表达在大鼠心肌缺血再灌注损伤模型和H_2O_2诱导H9c2氧化应激损伤模型中明显升高。H9c2细胞过表达Dp71通过提高Bcl2和lamin B1表达而抑制H_2O_2诱导的细胞凋亡。
OBJECTIVE: To establish a rat model of myocardial ischemia-reperfusion injury and oxidative stress injury, and to study the expression of dystrophin Dp71 in injured myocardium and cells, and to explore its molecular mechanism of antagonizing cardiomyocyte apoptosis. Methods: The left anterior descending coronary artery of SD rats was occluded for 30 minutes, then the model of myocardial ischemia-reperfusion injury was restored by blood flow. The changes of myocardial morphology, LDH and the apoptosis of cardiomyocytes were observed. The changes of Dp71 protein And m RNA expression. The oxidative stress injury model of H9c2 cells was established to detect the expression of Dp71 protein and m RNA in H 2 O 2 -stimulated cells. H9c2 cells were transfected with Dp71 overexpression plasmid. Flow cytometry was used to detect the effect of Dp71 overexpression on H_2O_2-induced apoptosis in H9c2 cells. Western Blot was used to detect the effect of Dp71 overexpression on the expression of lamin B1 and Bcl-2 protein and the effect of H_2O_2 on lamin B1 and Bcl2 protein expression. Results: Compared with the sham-operation group, the morphological changes of the HE-stained myocardium were observed in the reperfusion injury group, the LDH was significantly increased, and the number of apoptotic cardiocytes in the IR group was significantly increased. The mRNA and protein expression of Dp71 increased at each time point after IR (P <0.05). The expression of Dp71 protein and m RNA in cells stimulated with 0.2 mmol / L H 2 O 2 for 16 h was significantly increased (P <0.05). H9c2 cells transfected with Dp71 overexpression plasmid can inhibit H_2O_2 -induced apoptosis; H9c2 cells over-expression of Dp71 caused lamin B1 and Bcl2 expression increased, high expression of Dp71 can inhibit hydrogen peroxide-induced lamin B1 and Bcl2 expression decline. CONCLUSION: Dp71 m RNA and protein expression are significantly increased in a rat model of myocardial ischemia-reperfusion injury and H 2 O 2 -induced H9c2 oxidative stress injury. Overexpression of Dp71 in H9c2 cells inhibited H_2O_2-induced apoptosis by increasing the expression of Bcl2 and lamin B1.