摘要
目的冠状动脉微栓塞（coronary microembolization，CME）后导致的心肌凋亡是引起心功能损伤的主要原因之一，第10号染色体缺失的磷酸酶和张力蛋白同源物基因（phosphatase and tensin hmmlogydeleted on ten，PTEN）在CME后心肌凋亡中起到重要的作用，MicroRNA-21对心肌有保护作用，主要是通过调控其靶基因PTEN实现，因此本研究探讨超声微泡靶向转染MicroRNA-21对CME小猪心肌细胞凋亡的影响及意义。方法20头巴马小猪随机（随机数字法）分为假手术组、微栓塞组（CME组）、CME＋单纯基因组、CME＋超声介导基因组，每组小猪均为5只。经微导管左前降支注入微栓塞球构建CME模型组，注射等量生理盐水构建假手术组。CME＋超声介导基因组于CME前4天经耳缘静脉注入质粒一微泡混合液，同时予超声基因转染治疗仪经猪胸壁辐照心肌。CME＋单纯基因组于CME前4天经耳缘静脉注入质粒礅泡混合液，不予超声辐照。应用心脏超声检测心功能指标；组织病理切片HE及HBFP染色进行梗死区域测量；冰冻切片荧光显微镜评估绿色荧光蛋白（GFP）标记基因表达量；切口末端标记法检测心肌细胞凋亡；荧光定量PCR检测心肌组织PTENmRNA表达；Westernblot检测心肌组织PTEN与Caspase-3蛋白表达。结果（1）与单纯基因组比较，超声介导基因组可使心肌内外源基因表达水平提高8倍以上（P〈0．05）。（2）超声心动图参数显示，与假手术组[（67．87±2．36）％]比较，CME组[（50．94±3．52）％]和CME＋单纯基因组[（52．47±3．71）％]的左室射血分数（LVEF）显著降低（P〈0．05）；与CME组比较，CME＋超声介导基因组[（64．79±2．95）％]可改善CME导致的心功能损伤（P〈0．05）。（3）与假手术组比较，CME组PTEN的mRNA和蛋白表达量均显著增加，Caspase-3的蛋白表达量增加（均P〈0．05）；与CME组比较，CME＋超声介导基因组中PTEN的mRNA和蛋白表达量明显较低，Caspase-3的蛋白表达量较低（均P〈0．05）。结论超声微泡靶向转染MicmRNA-21可以有效地改善CME致心功能损伤，其可能机制是通过下调其靶基因PTEN在心肌细胞的表达，进而减少心肌细胞凋亡起效。
Objective To investigate the effects of ultrasound-targeted microbubble destructionmediated MicroRNA-21 on cardiomyocyte apoptosis after coronary microembolization （CME） in swine. Methods Twenty Bama miniature swine were randomLy （random number） divided into sham-operated, CME, CME plus gene transfection and CME plus ultrasound mediated gene transfection groups （ n = 5 per group）. The CME model was established by microcatheter-mediated injection of microspheres into the left anterior descending artery. The sham-operated group were made by injection of saline instead. The CME plus ultrasound mediated gene transfection group was made by injection of plasmid-microbubble mixture through the marginal ear vein 4 days before CME established. Meanwhile, ultrasound treatment was given to the myocardium through chest wall. The CME plus gene transfectian group was made by injection of plasmid- microbubble mixture through the marginal ear vein 4 days before CME established without exposure to ultrasound. Left ventricular ejection fraction （LVEF） was examined by cardiac ultrasound. Tissue biopsy was stained with hematoxylin-eosin （HE） and hematoxylin basic fuchsin picric acid （HBFP） to measure the size of infarction area. Green fluorescent protein （GFP） -labeled gene expression was evaluated by fluorescent microscopy in frozen sections. Cardiomyocyte apoptosis was detected with terminal deoxynucleotidyl transferase （TdT） -mediated dUTP nick end-labeling （TUNEL staining）. The expression of PTEN mRNA was measured by fluorescent quantitative PCR. The levels of PTEN protein and Caspase-3 protein was measured by western blot. Results ①Compared to CME plus gene transfection group, the CME plus ultrasound mediated gene group had over eightfold expression of exogenous genes in myocardium （P 〈 0. 05 ） measured by using optical density of green fluorescence protein; ②Compared with sham- operated group [ （67. 87 ± 2. 36） % ], the LVEF of CME group [ （50. 94 ± 3.52） % ] and CME plus gene transfection group [ （52.47±3.71）%] were markedly decreased （P 〈 0.05）. Compared with CME group, the CME plus ultrasound mediated gene transfection group [ （64. 79 ±2. 95） % ] improved CME- induced cardiac dysfunction as evidenced by increased LVEF （P 〈 0. 05） ; ③Compared with sham-operated group, the expression of PTEN mRNA and levels of PTEN protein and Caspase-3 protein in the CME group increased significantly （P 〈 0. 05 ）. Compared with CME group, the levels of PTEN protein and Caspase-3 protein and the expression of PTEN mRNA in CME plus ultrasound mediated gene transfection group was dramatically decreased （ P 〈 0.05 ）. Conclusions Ultrasound microbubble-mediated MicroRNA-21 transfection effectively improved CME-induced cardiac dysfunction by down-regulating the expression of targeted gene PTEN in myocardial cells, mainly reducing the post-CME myocardial cell apoptosis.
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2015年第7期712-718,共7页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金项目（81260042）
2014年广西研究生教育创新计划项目（YCBZ2014023）
