核定位信号肽在超声靶向破坏微泡介导基因转染治疗犬心肌梗死中的促进作用

摘要

目的 探讨超声靶向破坏微泡(UTMD)技术联合核定位信号(NLS)肽增强犬心肌hAng-1基因体内转染效率的可行性及应用价值.方法 构建犬急性心肌梗死模型并分组进行基因转染治疗,每组8只.A组:建模成功后未行治疗,即空白对照组;B组:建模成功后超声介导质粒DNA转染;C组:建模成功后超声介导质粒DNA加经典NLS肽转染;D组:建模成功后超声介导质粒DNA加突变NLS肽转染;E组:建模成功后超声介导质粒DNA加经典NLS肽和入核阻滞剂转染.建模后转染前,HE染色观察心肌梗死后病理变化情况.转染后3d,心肌冰冻切片检测EGFP标记的hAng-1基因表达情况,RT-PCR和Western Blot分别从基因和蛋白水平方面检测心肌组织hAng-1基因表达效率.转染后28 d,cα-SMA免疫组化定位新生毛细血管,并在显微镜下计数新生微血管密度;Masson染色及心脏大体标本评价心肌梗死区纤维化程度及面积.建模前、建模后1周基因转染前及基因转染后28 d分别应用二维超声心动图检测各组犬射血分数及室壁运动情况,比较各组基因治疗效果.结果 ①A、B、C组心肌组织中绿色荧光蛋白表达量逐渐增高,C、D、E组逐渐减少,Western Blot和RT-PCR检测结果显示hAng-1蛋白及基因表达趋势与绿色荧光蛋白一致,C组与其余各组比较差异均有统计学意义(均P＜0.05).(②免疫组化结果显示C组毛细血管密度最高,与其余各组比较差异均有统计学意义(均P＜0.05);Masson染色显示A、B、C组心肌纤维化程度依次减低,C、D、E组依次增高.③建模后1周,5组犬均可见节段性室壁运动减低且射血分数明显减低,组间比较差异无统计学意义.基因转染后28 d,A、E组心功能明显减低,B、D组轻度减低,C组轻度增高,C组与其余各组比较差异均有统计学意义(均P＜0.05).结论 UTMD联合NLS可高效、靶向地介导质粒DNA在缺血心肌内的表达,并促进血管新生,改善心肌纤维化,这种非侵入性的技术在心脏基因治疗方面将有较好的应用前景.%Objective To explore the feasibility and application value on enhancing gene transfection by ultrasound targeted microbubbles destruction (UTMD) combined with nuclear localization signal (NLS) petide in treating canine myocardial infarction.Methods Forty canines were randomly divided into 5 groups after the models of myocardial infarction prepared.Group A:blank control group(n=8),group B:UTMD+pDNA (n=8),group C:UTMD+pDNA+cNLS (n=8),group D:UTMD+pDNA+mNLS (n=8),group E:UTMD+pDNA+cNLS+WGA(n=8).Before transfection,HE staining was used to observe the pathological changes after myocardial infarction.3 d after gene transfection,the hAng-1 gene expression was detected in the frozen sections of the myocardium,RT-PCR and Western Blot were used to detect the expression of hAng-1 from the gene and protein level.28 d after gene transfection,α-SMA immunohistochemistry was used to detect capillary density of peri-infarct area and microvessel density (MVD) was detected.Masson's trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis.Echocardiography was used before myocardial infarction,one week after myocardial infarction and 28 d after gene transfection,the left ventricular ejection fraction(LVEF) and the left ventricular wall motion were detected.The gene transfection efficiency of each group were evaluated and compared.Results ①The expression of green fluorescence gradually increased from group A to group C,gradually decreased from group C to group E.Western Blot and RT-PCR showed that the hAng1 protein expression trends and the gene expression trends in 5 groups was consistent with the expression of green fluorescence.② Immunohistochemical showed the capillary density of group C was the highest,the differences of group C was statistically significant compared with other groups (all P＜0.05).Masson's trichromatic staining and cardiac gross specimen showed that the degree and area of myocardial fibrosis were reduced in an order of group A,B,C and increased in an order of group C,D,E.③One week after myocardial infarction,the left ventricular walls motion and LVEF in 5 groups were significantly decreased,there was no significant difference among the groups;28 d after gene transfection,the LVEF of group A and group E were significantly reduced,the group B and group D were slightly reduced,the group C was slightly increased.The differences of group C was statistically significant compared with other groups(all P＜0.05).Conclusion UTMD combined with NLS can be used to deliver plasmid DNA to the myocardium selectively and effectively,and it can promote neovascularization and improve myocardial fibrosis.This noninvasive technique is a promising method for cardiac gene therapy.