血管生成素1基因治疗抑制大鼠急性心肌梗死后心肌细胞凋亡和改善心脏功能

Intra-myocardial injection of Angiopoietin-1 plasmid inhibits cardiomyocyte apoptosis and improves cardiac function in rats with acute myocardial infarction

目的:观察心肌内直接注射血管生成素1基因对急性心肌梗死大鼠心肌细胞凋亡的影响,并进一步探讨血管生成素1基因治疗对急性心肌梗死后心室重构的影响。方法:实验于2004-09/2005-09在北京大学医学部生物化学与分子生物学基因组实验室完成。①选用SPF级近交系SD雄性大鼠95只,鼠龄6周,体质量(250±26)g。按随机抽签法将大鼠分为4组:假手术组(n=20)、模型组(n=28)、载体治疗组(n=24)、基因治疗组(n=23)。②结扎大鼠冠状动脉左前降支制备心肌梗死模型。假手术组:只进行前降支下穿线而未进行结扎。模型组:模型制备后于心肌梗死组织周围多点注射磷酸盐缓冲溶液。载体治疗组:模型制备后于心肌梗死组织周围多点注射载体质粒50μg。基因治疗组:模型制备后于心肌梗死组织周围多点注射50μg血管生成素1质粒。③术后3,7,14,28d进行心脏超声心动图检查。采用聚合酶链反应半定量分析外源血管生成素1mRNA和蛋白激酶B的mRNA表达水平。采用DNA片段原位末端标记法观察心肌细胞凋亡情况。④计量资料间差异比较采用单因素方差分析。结果:实验中因死亡等原因脱失15只,共有80只进入结果分析,各检测时间点各组分别为5只。①心肌组织内外源血管生成素1mRNA表达:术后3,7,14,28d,假手术组、模型组、载体治疗组均未见表达,基因治疗组均有表达,并维持在较高水平,注射后28d仍有表达。②心肌细胞凋亡数量:术后3,7,14,28d,基因治疗组心肌细胞凋亡数量均明显少于模型组和载体治疗组(P<0.05)。③心肌组织内蛋白激酶BmRNA表达:术后3,7,14和28d,基因治疗组明显高于模型组和载体治疗组(P<0.01),而模型组和载体治疗组差异不明显(P>0.05)。④心脏功能:术后7,14,28d模型组和载体治疗组的左心室舒张及收缩末期内径均明显大于假手术组和基因治疗组(P<0.05～0.01)。而基因治疗组术后7,14和28d心脏射血分数和左心室短轴缩短率明显高于模型组和载体治疗组(P<0.05)。结论:血管生成素1基因心肌内注射可能通过直接抑制心肌缺血时心肌细胞的凋亡,促进心肌细胞存活,延缓心肌梗死后的心室重构和心力衰竭的进展。

AIM： To observe the effects of intra-myocardial injection of plasmid angiopoietin-1 gene on myocardial cell apoptosis in rats with acute myocardial infarction, and further probe into the effects of angiopoietin-1 gene treatment on the remodeling of ventricles in rats with acute myocardial infarction. METHODS： The experiment was conducted in the Laboratory of Biochemistry and Molecular Biology, Medical Department of Peking University from September 2004 to September 2005.①Ninety-five SD male rats of SPF inbred strain aged 6 weeks old with the body mass of （250 ±26）g were selected and randomly divided into 4 groups： sham-operation group （n=20）, model group （n=28）, vector treatment group （n=24）, gene treatment group （n=23）.②Rats were ligated of the left anterior descending artery to made into acute myocardial infarction models. Sham-operation group： Rats were braided of the left anterior descending artery without ligation. Rats in the model group were injected with phosphate buffered solution at multiple points in the periphery of infarction tissues after model-establishment. Rats in the carrier treatment group were injected with 50 μg of vector plasmid at multiple points in the periphery of infarction tissues after model-establishment. Rats in the gene treatment group were injected with 50μg of angiopoietin-1 plasmid at multiple points in the periphery of infarction tissues after model establishment.③At 3, 7, 14, 28 days after operation, rats were examined by echocardiography. The polymerase chain reaction was used to semiquantitatively analyze the exogenous angiopoietin-1 mRNA and expression of mRNA in protein kinase B. The apoptosis of cardiomyocyte were studied by DNA segmental in situ end labeling method. ④The differences of measurement data were compared with single-factor analysis of variance. RESULTS： Fifteen rats withdrew from the experiment for death and others, and there were totally 80 rats involved in the analysis of results with 5 rats in each group at the detecting time-point.①The expressions of exogenous angiopoietin-1 mRNA in myocardial tissues： no expression was found in rats of sham-operation group, model group, cartier treatment group at 3, 7, 14 and 28 days after operation, while there were expressions in the gene treatment group, which was in a high level, and it could be found at 28 days after injection.②The number of apoptosis of cardiomyocyte： at 3, 7, 14 and 28 days after operation, it was significantly less in the gene treatment group than that in the model group and carrier treatment group （P 〈 0.05）. ③Expression of protein kinase B mRNA in the myocardial tissues： it was remarkably higher in the gene treatment group than that in the model group and cartier treatment group at 3, 7, 14, 28 days after operation （P 〈 0.01）, while there was no marked differences between the model group and carrier treatment group （P 〉 0.05）. ④Cardiac function： The dilation in left ventricle were obviously attenuated in the gene treatment group than that in the model group and vector treatment group at 7, 14 and 28 days after operation （P 〈 0.05）. At 3, 7, 14 and 28 days after operation, the ejection fraction and shortening fraction were remarkably lower in the model group and cartier treatment group than those in the sham-operation group （P 〈 0.01）, while those in the gene treatment group at 7, 14 and 28 days after operation were obviously higher than those in the model group and carrier treatment group （P 〈 0.05）. CONCLUSION： Intra-myocardial injection of Angiopoietin-1 plasmid may inhibit the apoptosis of cardiomyocyte in myocardial infarction, improve the survival of cardiomyocyte and ameliorate the ventricular remodeling of ventricle as well as the progress of heart failure after acute myocardial infarction bv activatinz the signal transduction oathwav of orotein kinase B.