超声靶向破坏微泡联合核定位信号肽增强基因转染治疗犬心肌梗死的实验研究  

Experimental study of combining ultrasound targeted microbubble destruction with nuclear localization signal peptide to enhance gene transfection in treatment of canines myocardial infarction

作　　者：崔晶晶[1] 周青[1] 曹省[1] 邓倾[1] 胡波[1] 王益佳[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,430060

出　　处：《中华超声影像学杂志》2017年第2期159-164,共6页Chinese Journal of Ultrasonography

基　　金：国家自然科学基金项目（81471674）;中央高校基本科研业务费专资金资助（2015301020203）

摘　　要：目的利用超声靶向破坏微泡（UTMD）技术联合核定位信号（NLS）肽提高人血管生成素-1（hAng-1）基因体内转染效率，治疗犬心肌梗死。方法46只犬经股动脉明胶海绵栓塞法成功建立急性心肌梗死模型后随机分为4组，每组10只：①空白对照（A）组；②hAng-1（B）组；③UTMD＋hAng-1（C）组；④UTMD＋NLS＋hAng-1（D）组。模型建成后1周，经静脉注入各组治疗试剂，同时C、D组超声辐照心前区。建模前、建模后1周基因转染前及转染后28d分别行超声检查，二维超声心动图检测各组犬射血分数及室壁运动情况，心肌声学造影检测各组犬梗死及周围区心肌血流灌注情况。转染后28d，RT-PCR和WesternBlot检测hAng-1基因相对表达量；免疫荧光SP法定位新生毛细血管，显微镜下计数新生毛细血管密度（MVD）；Masson染色及心脏大体标本评价梗死区纤维化程度及面积；比较4组基因治疗效果。结果①建模前，4组犬室壁运动、心功能正常，心肌造影各室壁未见充盈缺损。心肌梗死建模后1周基因转染前，4组犬室壁运动幅度及射血分数均明显减低，心肌造影均显示充盈缺损，组间差异无统计学意义（P〉0．05）。基因转染后28d，A、B、C、D组心功能依次增高，C、D组与其他各组比较差异有统计学意义（P〈0．05）。心肌造影D组可见较多造影剂充填，c组少许造影剂充填，A、B组造影剂充盈缺损。②RT-PCR和Western Blot显示D组hAng-1基因表达量明显高于其他组，C、D组与其他各组比较差异均有统计学意义（P〈0．05）。③免疫荧光sP法结果显示A、B、C、D组MvD依次增大，C、D组与其他各组比较差异均有统计学意义（均P〈0．05）。④Masson染色及心脏大体标本显示心肌纤维化程度及面积A-D组依次减低。结论NLS肽能促进UTMD介导hAng-1基因在缺血心肌内的高效表达，为缺血性心脏病的基因治疗提供�Objective To improve the canines myocardial infarction curative effect by using ultrasound targeted microbubble destruction （UTMD） combining with nuclear localization signal （NLS） peptide to increase hang-1 gene transfection efficiency. Methods Forty-six canines were randomly divided into 4 groups （ n = 10 in each group） after the models of myocardial infarction were prepared. Group A were untreated control; Group B were transfected with hAng-1;Group C were transfected with UTMD ＋ hang-1 ; Group D were transfected with UTMD ＋ NLS nuclear localization signal ＋ hang-1. The therapeutic agents were intravenously injected at one week after myocardial infarction in each group, and the ultrasound were irradiated at the precardium in group C and D. ①Echocardiography was used before and at one week after myocardial infarction and 28 days after gene transfection. Two-dimensional echocardiography was used to detect cardiac function, the left ventricular ejection fraction, the left ventricular wall motion and the myocardial contrast echocardiography were used to detect myocardial perfusion of all canines in the four groups. ②On twenty-eight days after gene transfection, mRNA and Western Blot were used to detect theexpression of hAng-1. Immunohistochemistry was used to detect capillary density of peri-infarct area and microvessel density （MVD）. Masson＇s trichromatic staining and gross specimen were used to evaluate the degree and the area of myocardial fibrosis. The gene transfeetion efficiency and the curative effect of all the four groups were evaluated and compared. Results ①Before myocardial infarction, in four groups canine ventrieular wall motion and cardiac function were normal, and myocardial filling defect was not showed by myocardial contrast echocardiography. At one week after myocardial infarction, the left ventricular anterior and interval walls motion and the left ventricular ejection fraction in the four groups were significantly decreased. Myocardial contrast eehoeardiography show

关 键 词：超声靶向破坏微泡 心肌梗死 基因治疗 核定位信号 

分 类 号：R445.1[医药卫生—影像医学与核医学] R542.22[医药卫生—诊断学]