| In modern life, the coronary artery disease has become the most important reason for disability and death. For now, there are several methods to treat coronary artery disease:medicine, PTC A and CABG, and so on. All of these treatment aim to rebuild tissue perfusion towards normal. However, current rebuilding methods are not suitable for refractory myocardial ischemia in those patients who have repeatedly myocardium infarcted. In recent years, with the development of gene therapy, therapeutic angiogenesis called "molecular bypass surgery" has been gradually payed attention to."Molecular bypass surgery" is to transfer angiogenesis gene or protein towards ischemic region to induce angiogenesis, restore the perfusion of the ischemic myocardium, activate the viable myocardium and hibernating myocardium, and then, as a result, improve the function of cardiac.The gene therapy for myocardial infarction (MI) has two questions to concern:the choice of the angiogenesis gene and the most suitable vector to transfer the therapeutic gene. Angiopoietion gene is a family of angiogenesis, mainly secreted by perivascular cells and vascular smooth muscle cells. In this family, Ang-1gene has multiple effects in angiogenesis. It can promote vessel wall’s maturation, reduce the leakage of blood vessel, and maintain the stability of new vessels. Ang-1took place later than VEGF right after infarction occurs, and act on different aspects of angiogenesis. Furthermore, in many aspects Ang-1can make up for deficiencies of VEGF, improve the reconfiguration of a failure heart. Therefore, the Ang-1gene has the potential of being a therapeuticangiogenesis gene in treatment of MI.Presently, the most clinically efficient method for gene transfection to specifictissue or organ to promote capillary generation remains unclear. Traditional viral and non-viral vector have various shortcomings, such as, high immunogenicity or low transfection efficiency. Another way of therapeutic angiogenesis is intramuscular administration, which is limited to its invasiveness. Ultrasound-mediated destruction of intravenously administered microbubbles has become a noninvasive method of promoted gene delivery administration. However, further research is required to improve the carrier’s target ability and gene transfection efficiency to achieve better biological effects. The bioeffect called "cavitation" and "sonoporation" can change the mobility of cell membrane and increase the membrane’s permeability. Therefore, ultrasound-mediated destruction of microbubbles carrying gene can make it easier for the target gene transfecting into target tissues and enhance the gene transfection efficiency as a result. Presently, however, the efficiency of gene transfection via ultrasound contrast agent through peripheral veins is not high enough for therapy. Some related clinical trials failed to display the same angiogenesis effect as animal trials due to the lack of target ability. Traditional transfection through intravenous injection has the possibility of causing serious systematic reactions yet.As a new carrier for gene transfection, ultrasound microbubbles’targeting can be achieved by the following methods:1)the microbubbles can be flashed by acoustic cavitation at the target tissue via ultrasonic irradiation;2) a specific antibody or ligand can be combined to the surface of the microbubbles to give specific adherence to the target tissue; and3) the therapeutic substance can be wrapped into the microbubbles and released through explosion by ultrasound at the target tissue.In case of myocardial ischemia, the injured endothelial cells excessively express multiple adherence molecules, mainly ICAM-1, while such expression is not obviously observed on the surface of normal endothelial cells. ICAM-1can mediate the combination of leukocyte and injured vascular, which can cause further damage on the vessels. Researches have demonstrated that the injured vascular intima could be adhered by large amount of microbubbles containing ICAM-1antibodies and therefore, this technology could be used for detecting the acute rejection of heart transplantation or injury of coronary artery. Hence, in this study, anti-ICAM-1antibodies were combined with microbubbles by the electrostatic attraction method to create targeted ultrasound contrast agents to recognize injured endothelial cells in the MI region. Part1hAng-1gene delivery by ultrasound-mediated SonoVue microbubbles to cultured293T cellObjective:To investigate the efficiency of hAng-1gene transfection to cultured293T cell using SonoVue microbubbles as vector mediated with ultrasound.Methods:Constructed eukaryotic expression plasmid pEGFP-C3-hAng-1. Cultured293T cells were divided into three groups:group A=hAng-1plasmid and microbubbles under ultrasonic irradiation; group B=hAng-1plasmid and ultrasound, without microbubbles; group C=hAng-1plasmid only (without ultrasound and microbubbles). Forty-eight hours after transfection, the transfection rate was detected by fluorescence microscopy and flow cytometry. RT-PCR and Western blot analysis were performed to evaluate the mRNA and protein expression of Ang-1, respectively. Cell viability was assessed by Trypan Blue staining. The integrity of the plasmid was evaluated by agarose gel electrophoresis.Results:The construction of the eukaryotic expression plasmid pEGFP-C3-hAng-1was successful which was confirmed by restriction enzyme maping analysis and DNA sequencing. Green fluorescence was observed in group A and B by fluorescence microscopy. The transfection efficiency was significantly improved in group A (P<0.01), compared with group B by flow cytometry. There was no significant green fluorescence observed in group C. The cell viability in group C was higher than those of group A and B. SonoVue microbubbles combined with ultrasound irradiation had no influence on the integrity of plasmid.Conclusions:Ultrasound irradiation along with SonoVue microbubbles couldn’t affect the integrity of pEGFP-C3-hAng-1plasmid. SonoVue microbubbles mediated with ultrasound could significantly improve the transfection efficiency of hAng-1gene to cultured293T cells.Part2SonoVue microbubbles carrying anti-ICAM-1antibodies targeted to injured endothelium in vitro and in vivoObjective:To explore the ability of SonoVue microbubbles carrying anti-ICAM-1 antibodies targeting to injured endothelium cells in vitro and vessel intima in vivo.Methods:Targeted SonoVue microbubbles carrying anti-ICAM-1antibodies were prepared in electrostatic attraction way with different volume ratio and pH value. The adherence rate of ultrasound agents and ICAM-1antibodies was detected by fluorescence microscopy and flow cytometry. The mixture of microbubbles and ICAM-1antibodies was added to IL-β stimulated ECV304cells which were divided into3groups as follow:group A:experimental group (stimulated ECV304cells and ICAM-1targeted microbubbles); group B:normal control group (normal ECV304cells with targeted microbubbles); and group C:negative control group (stimulated ECV304cells and ordinary microbubbles). Three acute myocardium infarction (AMI) rabbit models and normal rabbits were injected with microbubbles carrying ICAM-1antibodies or normal microbubbles intravenously to detect the ability of targeting in vivo.Results:The converted fluorescence microscope showed that the most appropriate volume ratio of dilution and pH value were1:50and4.0-5.0, respectively. Bright green fluorescence could be observed by converted fluorescence microscope in experimental group in vitro, which indicated that the targeted microbubbles carrying ICAM-1antibodies could combined to stimulated ECV304cells. There was only a little green fluorescence seen in the normal control group and no obvious green fluorescence in the negative control group. In vivo, luminous green fluorescence was observedon the injured vascular intima of the frozen cardiac muscle tissue sections in MI region in the experimental group. Very little green fluorescence was seen on the normal rabbits’ vascular intima. There was no obvious green fluorescence was observed in the MI rabbits which were injected with normal microbubbles. Similarly, no green fluorescence was observed in the hepatic and kidney tissue in rabbits.Conclusion:Targeted SonoVue microbubbles containing anti-ICAM-1antibodies could specifically recognize the injured vascular endothelium. Part3Directed hAng-1gene transfection by targeted ultrasound microbubbles containing anti-ICAM-1antibodies for the treatment of myocardial ischemia in rabbitsObjective:To explore the capability of hAng-1gene delivery in treatment of myocardial ischemia by ultrasound mediated targeted microbubbles carrying ICAM-1antibodies destruction.Methods:Forty-eight rabbits’left circumflex branch of coronary arteries were ligated for AMI models and were divided into5groups randomly as follow:the control group (C):without any treatment after the ligation; the U+P group:1ml hAng-1plasmid (with concentrate of100μg/ml in saline) was injected intravenously under ultrasound; the U+M+P group:1ml microbubbles and hAng-1plasmid (100μg/ml) were injected under ultrasound; the ICAM-1+UMP group (n=10):injected targeted microbubbles containing anti-ICAM-1antibodies and hAng-1gene under ultrasound; and the IM group:accepted direct intramuscular injection. Ultrasound cardiography and myocardial contrast echocardiography examination were performed on all animals before and2weeks after the treatment to evaluate the cardiac function and perfusion. All rabbits were executed2weeks after transfection and the gene transfection efficiency was detected by Real Time RT-PCR and Western blot analysis for hAng-1mRNA and protein expression, respectively. Microvessel density (MVD) counting of ischemic myocardium observed by Factor Ⅷ immunochemical staining was performed to value the proangiogenesis effect of hAng-1delivered by different administrations. Scar size of the myocardium after treatment was assessed by Evans Blue staining. The liver and the kidney tissue in U+P, U+M+P and ICAM-1group were taken to assess the systemic delivery.Results:There was significant improve of cardiac function and perfusion in the U+M+B group, ICAM-1+UMB group and IM group (P<0.01). Compared to the U+M+P group, the cardiac function had greater improvement in the ICAM-1+UMP and IM groups (P<0.05), which was similar between these two groups. The cardiac function and perfusion of the other two groups did not significantly increase. hAng-1mRNA and protein could be detected in U+M+P, ICAM-1and IM group, which was significantly higher in the last two groups (P<0.01), with no significant difference between them. None of the control animals showed hAng-1mRNA or protein expression. Compared with ICAM-1group, the MVD was greater in IM group (P<0.05), however. The hAng-1mRNA and protein were not detected either in liver or in kidney tissues in the three experimental groups with microbubbles. The scar size of the U+M+B group, ICAM-1+UMB group, and IM group decreased2weeks after the treatment, and was smaller in the ICAM-1+UMB group and IM group (P<0.01), which was not significantly different between the last two groups.Conclusions:Targeted SonoVue microbubbles containinganti-ICAM-1antibodies could deliver Ang-1gene to ischemic myocardium directly. Using ICAM-1targeted microbubbles as a gene vector was as effective as myocardium injection besides the greater proangiogenesis effect. This strategy had the ability and feasibility as a new gene carrier for myocardial ischemia. |
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