【作者】 任建丽；

【导师】 王志刚；

【作者基本信息】 重庆医科大学 ， 内科学， 2008， 博士

【摘要】 缺血性心脏病是影响人们生命健康的主要疾病之一,尽管国内外学者已经做了许多努力,但目前仍没有理想的治疗方法。近年来,随着基因重组技术的发展,基因治疗缺血性心脏病已成为研究的热点及方向,但外源基因导入人体组织细胞的安全性和有效性尚难以令人满意,也成为基因治疗应用于临床的主要障碍。研究发现,超声靶向破坏微泡(Ultrasound-targeted microbubble destruction, UTMD)所产生的机械效应和空化效应能够增加细胞膜的通透性,促进外源基因物质进入靶细胞。然而,在超声辐照下,载基因微泡破裂释放的基因只是被动地穿过细胞膜到达细胞内,进入胞浆、胞核的基因量及靶组织的基因转染效率限制了UTMD技术的广泛应用。穿膜肽具有较强的携带大分子物质的能力,它已经被证实能够转导大分子物质主动穿过细胞膜,与其他非病毒载体相比有许多优势,如与细胞膜亲和性高、穿膜速度快、可迅速被降解,最重要的是对细胞膜没有破坏性,为研制一种高效、安全的载体,主动介导核酸跨越生物屏障进行基因治疗提供了广阔的前景。如果将超声微泡与穿膜肽联合应用,既可使穿膜肽具有靶向性,又能提高载基因微泡的转染效率。因此,本课题进行了超声微泡联合穿膜肽促基因转染的体内、体外实验研究,观察基因转染效果及该技术介导HGF基因治疗急性心肌梗死的疗效,为超声靶向破坏微泡介导的基因治疗提供了一种新思路和新手段。本课?题研究主要包括以下二个部分的内容:第一部分超声微泡联合穿膜肽促基因转染的体外实验第一节TDL复合物介导基因转染的体外实验目的探讨HIV-1 Tat蛋白转导域/质粒DNA/Liposome (TDL)复合物介导增强型绿色荧光蛋白质粒(pEGFP)在体外培养人脐静脉内皮细胞(HUVEC)的转染效果。方法依据质粒DNA与Tat肽不同的电荷比分为8组,Lipofectamine 2000转染组作为对照组。将质粒DNA与Tat肽以不同的电荷比混匀,再加入2μl Lipofectamine 2000制成TDL复合物,琼脂糖凝胶电泳分析质粒DNA与Tat肽的结合力;荧光显微镜和流式细胞仪观察TDL复合物与lipofectamine 2000介导基因在体外培养HUVEC中的转染效果;MTT法检测TDL复合物对HUVEC生长活性的影响。结果所有TDL复合物组琼脂糖凝胶电泳均未发现明显DNA条带。Lipofectamine 2000转染组及所有TDL复合物组均可观察到绿色荧光,TDL复合物各组的绿色荧光均强于Lipofectamine 2000转染组(P<0.05)。Tat/DNA电荷比为8:1时,TDL复合物的转染效率最高,为22.03%,与其他各组比较差异均有统计学意义(P<0.05)。TDL复合物各组对细胞活性均无明显影响。结论TDL复合物可明显提高基因在体外培养HUVEC中的转染效率,并对细胞活性无明显影响。第二节超声微泡促进TDL复合物介导基因转染的体外实验目的探讨超声微泡促进TDL复合物介导的肝细胞生长因子(HGF)基因在HUVEC中的转染效果。方法将HUVEC接种在24孔板中,随机分为6组:①空白对照组;②TDL复合物组;③TDL复合物+微泡组;④TDL复合物+超声组;⑤TDL复合物+微泡+超声组;⑥质粒DNA+微泡+超声组。微泡终浓度为10%,超声辐照条件为1 MHz,0.75 W/cm2,辐照10 s,间隔10 s,共60 s。荧光显微镜及流式细胞仪检测pIRES2-EGFP-HGF在HUVEC中的表达及转染效率。MTT法检测该方法对HUVEC生长活性的影响。RT-PCR及Western Blot检测HGF的mRNA及蛋白在HUVEC中的表达。结果空白对照组没有观察到绿色荧光,其他各组均可观察到绿色荧光,TDL复合物+微泡+超声组的绿色荧光强度及转染效率均高于其他各组(P<0.05),并对细胞活性无明显影响。TDL复合物+微泡+超声组的HGF mRNA及HGF蛋白的表达也均高于其他各组(P<0.05)。结论超声微泡可增强TDL复合物介导的基因在体外培养HUVEC中的转染效率,并对细胞活性无明显影响,为缺血性心脏病的基因治疗提供了实验基础。第二部分超声微泡联合穿膜肽促基因转染治疗急性心肌梗死的实验研究第一节穿膜肽Tat的固相合成及体外活性研究目的以固相法合成穿膜肽Tat,并对合成产物进行活性评价。方法采用9-芴甲氧羰基(Fmoc)作为α-氨基的保护基,以逐个延伸的固相法合成穿膜肽Tat。应用高效液相色谱和质谱仪测定其纯度及分子量;荧光显微镜观察穿膜肽Tat介导pEGFP在体外培养HUVEC中的转染效果以评价穿膜肽Tat的生物活性;MTT法检测穿膜肽Tat与质粒DNA复合物对HUVEC生长活性的影响。结果高效液相色谱和质谱鉴定所合成穿膜肽Tat的纯度为96.6%,分子量为1 880。它能够携带质粒DNA穿过细胞膜进行基因转染,并对细胞活性无明显影响。结论采用Fmoc固相合成法可以成功地合成有生物活性的穿膜肽Tat。第二节载基因及穿膜肽脂质超声造影剂制备的实验研究目的研究自制的脂质超声造影剂载基因及穿膜肽的能力,评价其物理性质、显影效果及体内转染情况。方法采用机械振荡法制备载基因及穿膜肽的脂质超声造影剂,光学显微镜观察其大小及分布;马尔文测量仪测其粒径及表面电位;血球计数仪在光镜下测定其浓度;激光共聚焦显微镜观察质粒DNA和穿膜肽Tat在超声造影剂中的分布;荧光分光光度仪测定微泡中基因和穿膜肽的包封率;DFY-Ⅱ型超声图像定量分析诊断仪定量分析造影前后的图像;超声基因转染治疗仪进行体内转染,采用激光共聚焦显微镜观察pIRES2-EGFP-HGF在大鼠心肌中的表达。结果自制载基因及穿膜肽脂质超声微泡造影剂大小分布均匀,分散度好,粒径为(2.27±0.38)μm,浓度为(3.07±0.42)×109个/ml,表面电位为(1.95±0.13)mv。质粒DNA和穿膜肽均粘附于微泡表面并包裹入微泡内。该造影剂中基因的包封率为32 %,穿膜肽的包封率为35 %。体内造影实验显示心腔和心肌造影后的回声强度值均高于造影前(P<0.05)。体内转染实验显示载基因及穿膜肽的脂质超声造影剂可明显增强基因的转染效率。结论自制载基因及穿膜肽脂质超声造影剂符合普通超声造影剂的要求,载基因及穿膜肽的效率高,能增强心肌显影及基因转染效率,可作为携带基因等生物活性物质的载体材料。第三节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(AMI)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+Tat-MB),④超声+载基因微泡组(US+HGF-MB),⑤超声+载基因及穿膜肽微泡组(US+HGF-Tat-MB)。心梗模型建成后3 d,经股静脉注入超声微泡,同时超声基因转染治疗仪辐照心前区,辐照条件为300 KHz,2 W/cm2,辐照5 s,间隔5 s ,共6 min。于超声辐照后7 d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型、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及蛋白在大鼠心肌中的表达。结果US+HGF-Tat-MB组的绿色荧光强度高于其他各组,I型、III型胶原明显少于其他各组。IHC结果显示新生的微血管被染成棕黄色,US+HGF-Tat-MB组的MVD最高,为(142.40±7.99)个/HP,与其他各组比较差异均有统计学意义(P<0.01)。US+HGF-Tat-MB组的HGF mRNA及HGF蛋白的表达均高于其他各组(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表达,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。更多还原

【Abstract】 Ischemic heart disease is a major global health problem. Although great efforts have been made in the past few years, no ideal treatment is available. Gene therapy shows considerable promise as a new modality for ischemic heart disease. The progress of gene therapy largely depends on the development of gene delivery technologies, including viral vector and non-viral vector. However, the development of gene therapy is still slow mainly because of the safety of viral vector and poor efficiency of non-viral vector. Therefore, an important goal in gene therapy area is to develop a novel and efficient gene delivery system. Recent studies showed that mechanical and cavitation effects caused by ultrasound-targeted microbubble destruction (UTMD) were able to increase membrane permeability and enhance exogenous genetic materials into targeted cells. However, gene released from microbubbles destructed by ultrasound radiation only passively permit into targeted cells, the amounts of gene in endochylema and nucleus and gene transfection efficiency in targeted tissue limit the extensive application of UTMD. In recent years, cell-permeable peptides (CPPs) have been widely used as cellular delivery vectors for its remarkable functions of delivering macromolecular substance directly and actively through cellular membrane into endochylema or nucleus without cytotoxicity, and have high affinity of cellular membrane, fast speed of permeating membrane, fast degradation and no destructiveness to cellular membrane. Transactivating transcriptional activator protein from human immunodeficiency virus type 1(HIV-1 Tat), one of most popular CPPs, owns membrane translocation property and non-cytotoxicity.Combination with ultrasound microbubbles and Tat peptide, not only Tat peptide has the targeting, but also the transfection efficiency of microbubbles carrying gene was enhanced. The experiment of combination of ultrasound microbubbles and Tat peptide in vitro and in vivo was done, and observed the effect of gene transfection and therapeutic effect of the technique mediated HGF gene treating acute myocardial infarction. It would provide a new thought and a new method for gene therapy mediated by UTMD. The study included the following two parts. PARTⅠ: Combination of ultrasound microbubbles and Tat peptide enhanced gene transfection in vitroSECTIONⅠ: Tat peptide/plasmid DNA/liposome (TDL) compound mediated gene transfection in vitroObjective To investigate the transfection efficiency of plasmid vector coding enhanced green fluorescence protein (pEGFP)in human umbilical vein endothelial cell (HUVEC) using Tat peptide/Plasmid DNA/Liposome compound (TDL compound). Methods Plasmid DNA was mixed with Tat peptide by various charge ratio, then added 2μl Lipofectamine 2000 to form TDL compound. Bonding force of plasmid DNA and Tat peptide was analyzed by agarose gel electrophoresis. Transfection effect of TDL compound and Lipofectamine 2000 in HUVEC was observed using fluorescent microscopy and flow cytometry (FCM). The viability of HUVEC was measured by MTT assay. Results A visible strap was not found in all group of TDL compound by agarose gel electrophoresis. Transfection efficiency of TDL compound was higher than that of Lipofectamine 2000 (P<0.05). When charge ratio of Tat and DNA was 8:1, transfection efficiency of TDL compound was highest. No significant difference of cell viability was found in all TDL groups. Conclusion TDL compound can enhance the efficiency of gene transfection without obvious damage to cell viability in HUVEC, which might become an effective means of enhancing the efficiency of gene transfection, and it offers a new strategy to increase the transfection efficiency of non-viral genetic vector.SECTIONⅡ: Transfection efficiency of TDL compound in HUVEC enhanced by ultrasound-targeted microbubble destructionObjective To explore the gene transfection efficiency of TDL compound combined with UTMD in HUVEC. Methods Tat peptide, plasmid DNA (pIRES2-EGFP-HGF) and Lipofectamine 2000 were used to prepare the TDL compound. Microbubbles were prepared using mechanic vibration. The cells were seeded in 24-well plates, which were randomly assigned into six groups.①blank control group,②TDL compound group,③TDL compound + microbubbles group,④TDL compound + ultrasound group,⑤TDL compound + microbubbles + ultrasound group,⑥plasmid DNA + microbubbles + ultrasound group. The expression of the report gene pEGFP was observed using fluorescent microscopy and FCM. The viability of HUVEC was measured by MTT assay. mRNA and protein of HGF was analyzed by RT-PCR and Western Blot. Results The intensity of green fluorescence and the gene transfection efficiency of TDL compound + microbubbles + ultrasound group were higher than those of other groups, and no significant difference of cell viability was found between TDL compound + microbubbles + ultrasound group and the other groups. The HGF mRNA and HGF protein of TDL compound + microbubbles + ultrasound group were higher than those of other groups.Conclusion Our finding demonstrated that UTMD could enhance the transfection efficiency of TDL compound without obvious effects on the cell viability of HUVEC, suggesting that the combination of UTMD and TDL compound might be a useful tool for the gene therapy of ischemic heart disease.PARTⅡ: Combination of ultrasound microbubbles and Tat peptide enhanced HGF gene transfection to treat acute myocardial infarction SECTIONⅠ: Solid synthesis and bioactivity in vitro of Tat peptideObjective To synthesize Tat peptide by the solid-phase synthetic method and evaluate the bioactivity. Methods Tat peptide synthesis was performed by the stepwise solid-phase method using the Fmoc group for protecting theα-amino acid. The purity and molecular weight of Tat peptide were determined using HPLC and MALDI-TOF-MS. Transfection effect of Tat peptide-mediated pEGFP in HUVEC was observed using fluorescent microscopy. Cell viability was determined by MTT assay. Results Peptide synthesized was verified using HPLC and MALDI-TOF -MS. The purity of Tat peptide was 96.6%, and molecular weight of Tat peptide was 1 880, and Tat peptide significantly increased the expression of reporter gene pEGFP in HUVEC, and no significant difference of cell viability was found. Conclusion Tat peptide was successfully synthesized by Fmoc solid-phase synthetic method and showed the bioactivity enhancing gene transfection.SECTIONⅡ: A novel ultrasound microbubbles carrying gene and Tat peptide: preparation and characterizationObjective To prepare a novel lipid ultrasound microbubbles carrying gene and Tat peptide,investigate the physical characterization of the microbubbles carrying gene and Tat peptide , its contrast-enhanced ultrasonography and transfection effect in vivo. Methods The lipid ultrasound microbubbles were prepared using mechanical vibration. The appearance, distribution, concentration, diameter and zeta potential of the lipid ultrasound microbubbles were measured. The efficiencies of the microbubble carrying gene and Tat peptide were investigated using confocal laser scanning microscopy and fluorospectrophotometer. The contrast-enhanced ultrasonography was performed on six normal rabbits to observe the duration and intensity of enhancement in the heart chambers and myocardium after the injection of the above microbubbles. Quantitative analysis was detected using a sonogram quantitative assay systems. Transfection in vivo was performed using an ultrasound gene transfection treatment meter. The expression of pEGFP in the rat myocardium was observed using confocal laser sanning microscope. Results The diameter of the lipid microbubbles carrying gene and Tat peptide was (2.27±0.38)μm, the concentration was (3.07±0.42)×109 /ml and Zeta potential was (1.95±0.13) mv. The gene encapsulation efficiency of the lipid ultrasound microbubbles was 32 %, and the Tat encapsulation efficiency was 35 %. The in vivo experiment showed that the novel lipid ultrasound microbubbles could significantly enhance the echo intensity and transfection efficiency. Conclusion The efficiency of the novel lipid microbubbles carrying gene and Tat peptide was high, which could be used as a new vehicle delivering genes or drugs for therapy as well as a potential ultrasound contrast agent.SECTIONⅢ: Ultrasound-targeted microbubble destruction using microbubbles carrying gene and Tat peptide enhanced HGF gene delivery to the infarcted myocardium in ratsObjective To explore the feasibility of mediated HGF gene treating acute myocardial infarction by UTMD using microbubbles carrying gene and Tat peptide. Methods Forty Sprague-Dawley rats were randomly divided to 5 groups after the models of myocardial infarction were prepared.①blank control group(C),②ultrasound+blank microbubbles group (US+MB),③ultrasound+loaded-Tat-microbubbles group (US+Tat-MB),④ultrasound+loaded-HGF-microbubbles group (US+HGF-MB) and⑤ultrasound+loaded-HGF-Tat-microbubbles group (US+HGF-Tat-MB). All the rats were sacrificed 7 days after gene transfection. The expression of pIRES2-EGFP-HGF in the rat myocardium was observed using confocal laser microscopy. Distribution of collogen in the rat myocardium was observed using polarizing microscopy. The CD34 expression was detected by IHC, and microvessel density (MVD) was deternined. The expression of HGF mRNA and HGF peptide was respectively detected by RT-PCR and Western Blot. Results Green fluorescence intensity of US+HGF-Tat-MB group was higher than those of other groups, its collogen was fewer than those of other groups, and its expression of HGF mRNA and HGF peptide was higher than those of other groups. IHC showed that MVD of US+HGF-Tat-MB group was the highest among all the groups (142.40±7.99/HP vs 40.00±3.39/HP, 78.40±3.97/HP, 80.80±3.35/HP, 112.20±5.81/HP). Conclusion UTMD using microbubbles carrying gene and Tat peptide could transfect HGF gene effectively and noninvasively to the impaired myocardium, promote angiogenesis and improve fibrosis, which providing a novel strategy for gene therapy of ischemic heart disease.更多还原