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  

The enhanced effect of nuclear localization signal petide on treating canine myocardial infarction with ultrasound targeted microbubbles destruction mediated gene transfection

作　　者：崔晶晶[1] 曹省[1] 陈金玲[1] 邓倾[1] 王益佳[1] 周青[1] 胡波[1] 

机构地区：[1]武汉大学人民医院超声影像科心血管病湖北省重点实验室,武汉市430060

出　　处：《临床超声医学杂志》2017年第1期1-6,共6页Journal of Clinical Ultrasound in Medicine

基　　金：国家自然科学基金项目(81471674);国家青年科学基金项目(81501495)

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h Ang-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3 d,心肌冰冻切片检测EGFP标记的h Ang-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h Ang-1基因表达效率。转染后28 d,α-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 (1)A、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h Ang-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＋p DNA（n=8）,group C：UTMD＋p DNA＋c NLS（n=8）,group D：UTMD＋p DNA＋m NLS（n=8）,group E：UTMD＋p DNA＋c NLS＋WGA（n=8）.Before transfection,HE staining was used to observe the pathological changes after myocardial infarction.3 d after gene transfection,the h Ang-1 gene expression was detected in the frozen sections of the myocardium,RT-PCR and Western Blot were used to detect the expression of h Ang-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（1） The 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 h Ang-1 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 P0.05）.Masson＇s trichromatic staining and cardiac gross specimen showed that the degree and area of myoca

关 键 词：非病毒载体 超声靶向破坏微泡 核定位信号 基因治疗 心肌梗死 犬 

分 类 号：R542.2[医药卫生—心血管疾病] R540.45[医药卫生—内科学]