Experimental Study Of Gene Transfer And Targeting Adherency By Ultrasound-mediated SonoVue Microbubble Destruction In Vitro

BackgroundIn recent years, with the development of recombinant gene technology, a molecular lever treatment of ischemic heart disease that "molecular bypass surgery" has gradually received attention. "Molecular bypass surgery" has been called "therapeutic angiogenesis". The most current research method of therapeutic angiofenesis is transfect gene into the cardiovascular tissue by gene vectors. However, traditional viral vector and non-viral vector have various shortcomings, such as, unsafety and low transfection efficiency. These shorcomings severely affected the clinical efficacy of gene therapy. So the establishment and development of efficient, safe, controllable, simple and practical gene gene therapy vector system has become the focus of the study. Recent studies show that ultrasound irradiation can promote gene transfer in vitro or in vivo, and ultrasound-mediated microbubble destruction can significantly increase gene transfection efficiency.Currently, the ultrasound contrast agents are containing gas tiny bubbles. The shell materials are phospholipids, albumin, sugar, non-ionic surfactant or biodegradable polymer and so on. The gas in the bubbles includes carbon dioxide, oxygen, air or large inert gas molecules (mostly halothane gas) and so on. As the contrast agent proteins or cationic liposome shell is usually positively charged, while the plasmid DNA is usually with a large number of negative charges, so the gene can adhere to the microbubble surface. In addition, the lipid shell microbubbles can also assemble different ligands, such as antibodies antibody Fab segment, amino acids or polysaccharides, to the microbubbles surface, which was prepared with tissue-specific targeting of microbubbles created the possibility. SonoVue microbubbles contain sulfur hexafluoride gas, and lipid shell. Microbubbles average diameter are 2.5μm.Large amounts of data shows that ultrasound can increase cell membrane permeability, and the destruction of microbubbles can break in the microvascular and widen the endothelial cell gap. Bao suggested that the main mechanism for these effects is the ultrasonic "cavitation". The presence of microbubbles can also reduce the ultrasonic cavitation threshold. Therefore, in target tissue, ultrasound irradiates the microbubbles carrying gene and the cavotation can make it easier for the target gene into tissues, to enhance the gene transfection and expression.The method of microbubble carrying the gene are mainly four kinds:①genes to microbubble surface or embedded in the suface;②penetrating foam integrated within the gene;③through the non-covalent binding adhesion gene to microbubble surface by electrostatic interaction;④by adding a layer of oil ester surrounding the microbubble, and then integrated into the hydrophobic drugs in this layer of oil ester. The first two methods currently used to achieve the target gene with the microbubble connection.The targeted microbubbles are the basis of targeted ultrasound molecular imaging, and the key technology of preparating targeted microbubbles is of surface modification of microbubbles. Connection methods commonly have covalent methods or non-covalent methods.ICAM-1 is a kind of cell adhesion molecule, a member of immunoglobulin family, mainly expressed on activated endothelial cells and other antigen transmitter cells.ICAM-1 mediated adhesion response of endothelial cells and leukocyte. Inflammatory cytokines induced upregulation of ICAM-1 and the latter mediated the leukocyte and endothelial cell interactions that is a key factor of inflammatory response. After ischemic a variety of inflammatory cytokines induced microvascular endothelial cells to express ICAM-1 and increased its level.And damaged endothelial cells exposed to a number of binding sites for inflammatory cells, such as ICAM-1 locus.Microbubbles can bind with these sites of inflammatory cells. Since ICAM-1 almost has not expression in normal tissues, labeled anti-ICAM-1 targeted microbubbles can specifically bind to damaged endothelial surface.Ang-1 gene is a family of angiogenesis factors, mainly secreted by perivascular cells, vascular smooth muscle cells and tumor cells. Ang-1 has multiple roles in angiogenesis. It can promote blood vessel maturation, reduce blood vessel leakage, and maintain the stability of blood vessels. In addition, Ang-1 can bind to the specific receptors Tie-2 on the endothelial cell and have a strong role of specific chemokines and migration of endothelial cells. Thus, Ang-1 and VEGF act on different aspects of angiogenesis, and in many areas Ang-1 can make up for deficiencies of VEGF. Part 1 Construction of recombination eukaryotic expression vector pEGFP-C3-hAng-1Objective To construct a eukaryotic expression vector containing the enhanced green fluorescence protein and recombinant human Angiopoietin-1.Method The HindⅢand BamH I primers specific for amplifying the DNA fragment encoding hAng-1 were designed and synthesized. The targeted DNA fragment was obtained from human totally RNA by RT-PCR. The pEGFP-C3 vector had been enzyme digestion by BamH I. Cutting pEGFP-C3 vector and hAng-1 with HindⅢand BamH, and using T4 ligase to connect the pEGFP-C3 vector and hAng-1. The recombinant plasmid pEGFP-C3-hAng-1 was first propagated in E.coli 5a, and then was confirmed to contain hAng-1 cDNA sequence by agarose gel electrophoresis and DNA sequence analysis.Result The construction of the recombinate eukaryotic expression plasmid pEGFP-C3-hAng-1 and the correction of the open reading frame were confirmed through restriction enzyme maping analusis and DNA sequencing.Conclusion By gene recombinant technology, the hAng-1 gene can be cloned into pEGFP-C3 vector to construct the recombinant eukaryotic expression plasmid pEGFP-C3-hAng-1.Part 2 Effects of ultrasound-mediated SonoVue microbubbles destruc-tion on the integrity and expression of hAng-1 geneObjective This study was desigend to test the efficiency of gene transfer and expression mediated by ultrasound and microbubble strategy in 293T cell.Methods Sonove microbubbles were mixed with plasmid DNA encoding hAng-1 and green fluorescent protein. The mixture of the DNA and microbubbles was administered to cultured 293T cells by ultrasound exposue. Forty eight hours later, transfer rate was assessed by fluorescence microscopy and flow cytometry. Cell viability was assayed by Trypan Blue staining. RT-PCR and Western blot analysis was used to examine the expression of hAng-1 mRNA and protein. Agarose gel electrophoresis was used to evaluate the integrity of the plasmid.Result The transfection expression rate of pEGFP in 293T cells was markedly increased with the ultrasound condition of 1.5w/cm2 and 30s, the additon of 20% microbubbles and 15μg/ml DNA. Fetal calf serum had no influence on the gene transfer rate. Ultrasound irradiation combined with microbubbles couldn’t destroy the integrity of plasmid.Conclusion Ultrasound-mediated microbubbles destruction can increase the transfection and expression of hAng-1 gene in 293T cells.Part 3 Experimental study on detecting damaged endothelium with the targeted mirobubbles carrying ICAM-1 antibodyObjective To discuss the feasibility of targeted microbubbles conjugated to ICAM-1 antibody in evaluating damaged endothelium.Methods Targeted microbubbles carrying ICAM-1 antibody was prepared by the electrostatic attraction way. Targeted microbubbles carrying ICAM-1 antibody was added to activate ECV304 in vitro. In vivo, the target ability of targeted microbubble to the damaged endothelium was detected.Results The results of homing test in vitro showed that the targeted microbubbles carrying ICAM-1 antibody could be combined with ECV-304 stimulated by IL-1βspecifically. The targeted microbubbles carrying antibody retained within the damaged endothelium were much more than the standard endothelium.Conclusion Microbubbles conjugated to ICAM-1 have been shown to bind to damaged endothelial cell.