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Combination Of Ultrasound Microbubbles And Tat Peptide Enhanced HGF Gene Transfection To Treat Acute Myocardial Infarction

Posted on:2009-12-14Degree:DoctorType:Dissertation
Country:ChinaCandidate:J L RenFull Text:PDF
GTID:1114360245488661Subject:Internal Medicine
Abstract/Summary:PDF Full Text Request
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.
Keywords/Search Tags:Ultrasonograph, Ultrasound contrast agent, Tat peptide, Hepatocyte growth factor, Myocardial infarction
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