摘要
目的探讨超声破坏微泡造影剂在体外及体内介导Ang-1基因转染的效率及其促血管新生的作用。方法293T细胞悬液分为3组：A组（微泡＋超声＋质粒组），B组（单纯超声照射＋质粒组）及C组（对照组，每孔仅加入目的基因质粒）；转染后48h用荧光显微镜和流式细胞仪分别定性、定量检测基因的转染效率，PCR及Western-blot分别检测细胞转染后的目的基因表达与蛋白翻译。27只健康成年中国家兔行急性前壁心肌梗死造模后随机分为3组：组Ⅰ（超声辐照＋微泡＋质粒组）、组Ⅱ（超声辐照＋质粒组）、组Ⅲ（对照组，心肌梗死后不做任何处理）；于基因转染前后分别行心肌超声造影（MCE）检查，并于2周后处死取心肌组织行PCR及Westernblot检测Ang-1mRNA及其蛋白的表达，心肌组织Ⅷ因子染色检测其新生毛细血管密度。结果48h后可在荧光显微镜下观察到A组及B组转染成功的细胞胞浆内发出绿色荧光，其中c组未见明显绿色荧光表达，A组的绿色荧光表达量明显高于B组；流式细胞仪检测A组转染效率较B组显著提高（P〈O．01）。体内转染中，心肌超声造影可见组Ⅰ转染前充盈缺损处出现片状造影剂回声，PCR可检测出Ang-1mRNA表达，Westernblot可检测出目的基因的蛋白产物，余各组则无明显造影剂充填，PCR、Westernblot均无法检测出Ang-1基因及其蛋白的表达；心肌毛细血管计数显示组Ⅰ新生毛细血管数量显著提高，余各组仅见少量新生毛细血管。结论超声破坏微泡造影剂可明显提高Ang-1基因的体外及体内转染效率，具有良好的促血管新生作用。
Objective To investigate the transfection efficacy and expression of Ang-1 gene and proangiogenesis in vitro and vivo by ultrasound-mediated microbubhle destruction. Methods 293T cells were divided into three groups： group A was given hAng-1 plasmid and microbubbles plus ultrasonic irradiation,group B was given hAng-1 plasmid and ultrasound, group C was given hAng-1 plasmid only （without ultrasound）. Forty-eight hours after transfection,the transient expression rate was observed under fluorescence microscopy and flow cytometry. RT-PCR and Western blot analysis were taken to evaluate the mRNA and protein expression of Ang-1 respectively. Twenty-seven rabbit models of ligated left circumflex branch coronary artery were divided into 3 groups randomly as follow： group I （accepted intravenous injection of SonoVue microbubble and Ang-1 plus ultrasonic irradiation）, group II （accepted intravenous injection of Ang-1 with ultrasound）, group III （control group）. Myocardial contrast echocardiography （MCE） was executed on all animals before and after the treatment. Two weeks after gene delivery, RT-PCR and Western blot analysis were taken to evaluate mRNA and protein expression of Ang-1 respectively. Microvessel density （MVD） counting of infracted myocardium, observed by Factor immunochemical staining, was performed to value the proangiogenesis effect of Ang-1 delivered by ultrasound mediated cavitation of microbubble. Results Green fluorescence was observed in group A and B by fluorescence microscopy,which was negative in group C. The transfection expression rate was significantly improved in group A （P 〈0.01）. In vivo, Microbubbles could be observed in former ischemic myocardium in MCE examination and the Angl mRNA and protein could be detected in group I. On the other hand,the contrast agent was defected obviously and none of the animals showed Ang 1 mRNA and protein expression in other two groups. The MVD counting showed significant improvement in group I whereas other two groups didn�
出处
《中华超声影像学杂志》
CSCD
北大核心
2012年第1期65-70,共6页
Chinese Journal of Ultrasonography
基金
国家自然科学基金面上项目（30600141）
关键词
超声检查
微气泡
转染
血管生成素1
Ultrasonography
Microbubbles
Transfection
Angiopoietin 1
