超声微泡联合穿膜肽促基因转染治疗急性心肌梗死的动物实验研究  

Experimental Research on Treating Acute Myocardial Infarction by the Combination of Ultrasound Microbubbles and TAT Peptide Enhancing Gene Transfection

作　　者：任建丽[1] 张萍[1] 周志益[2] 李攀[2] 王志刚[2] 

机构地区：[1] 重庆医科大学附属第二医院超声科,重庆市400010 [2] 重庆医科大学附属第二医院超声影像学研究所,重庆市400010

出　　处：《中国超声医学杂志》2014年第4期364-368,共5页Chinese Journal of Ultrasound in Medicine

基　　金：国家自然科学基金青年科学基金项目(No.81000621);教育部博士点(新教师类)基金项目(No.20105503120008)

摘　　要：目的探讨超声微泡联合穿膜肽介导HGF基因治疗大鼠急性心肌梗死的可行性。方法将40只SD大鼠建成心肌梗死模型后随机分为5组,即①空白对照组(C),②超声+空白微泡组(US+MB),③超声+载穿膜肽微泡组(US+MB_(TAT)),④超声+载基因微泡组(US+MB_(HGF)),⑤超声+载基因及穿膜肽微泡组(US+MB_(HGF+TAT))。于基因转染后7d处死各组大鼠,激光共聚焦显微镜观察pIRES2-EGFP-HGF在大鼠心肌内的表达情况。偏振光显微镜观察Ⅰ、Ⅲ型胶原在大鼠梗死心肌周边区的分布情况。免疫组织化学法(IHC)检测梗死心肌周边区CD34的表达,并在显微镜下计数心肌内新生微血管密度(MVD)。RT-PCR及Western Blot检测HGF的mRNA及蛋白的表达。结果 US+MB_(HGF+TAT)组的绿色荧光强度高于其他各组,Ⅰ、Ⅲ型胶原明显少于其他各组,HGF mRNA及HGF蛋白的表达均高于各组,IHC结果显示新生的微血管被染成棕黄色,US+MB_(HGF+TAT)组的MVD最高,与其他各组比较差异均有统计学意义(P<0.05)。结论超声微泡联合穿膜肽能够介导HGF基因在缺血心肌内的高效转染,并促进血管新生和改善纤维化,为缺血性心脏病的基因治疗提供一种新的基因转移途径。Objective To explore the feasibility of mediated HGF gene treating acute myocardial infarction by the comblnation of ultrasound microbubbles and TAT peptide. Methods Forty SD rats were randomly divided into 5 groups after the models of myocardial infarction were prepared, （1）blanked control group（C）, （2）ultrasound＋ blanked microbubble group （US＋ MB）, （3） ultrasound ＋ TAT-loaded microbubble group （ US ＋ MBTAx ）, （4） ultrasound ＋ HGF- loaded microbubble group （ US ＋ MBHGF ） and （5） ultrasound ＋ HGF-TAT-loaded microbubble group （ US ＋ MBHGF＋TAT）. All the rats were killed 7 days after gene transfection. Expression of the report gene EGFP in the rat myocardium was observed using laser eonfocal microscopy. Distribution of collagen in the rat myocardium was ob- served using polarizing microscopy. The CD34 expression was detected by IHC, and microvessel density （MVD） was deternined. Expression of HGF mRNA and HGF protein was detected respectively by RT-PCR and Western Blot. Re- suits Green fluorescence intensity of US＋MBHGF＋TAT group was higher than that of other groups, its collagen was fe- wer than that of other groups, and its expression of HGF mRNA and HGF protein was higher than that of other groups. IHC results showed that MVD of US＋ MB.GF＋TAT group was the highest among the groups. Conclusions The combination of ultrasound microbubbles and TAT peptide could effectively deliver HGF gene into isehemic myo- cardium, promote angiogenesis and improve fibrosis, which could provide a novel strategy for gene therapy of ischemic heart disease.

关 键 词：超声微泡 穿膜肽 基因转染 心肌梗死 

分 类 号：R542.22[医药卫生—心血管疾病] R445.1[医药卫生—内科学]