Combination Of Ultrasound Microbubbles And Recombinant Asenovirus AD-EGFP/HIF-1Α Mediate EPCS To Rat Myocardial Infarction

Ischemic heart disease (IHD) is one of the major serious global health problems. Although coronary heart disease (CHD) mortality has decreased with the extensive application of drugs,percutaneous coronary intervention and coronary artery bypass graft, a number of patients are still not suitable for those treatments. Myocardial angiogenesis is the focal and difficult point of the current studies of ischemic heart disease. In recent years, researchers treated ischemic heart disease by using stem cells transplantation, which become a hot spot direction. The current studies most focus on mononuclear cells (MNCs) and mesenchymal stem cells (MSCs). But with the increased number of cases in clinical application, reports of adverse reaction are also increased, especially arrhythmias, etc.Endothelial progenitor cells (EPCs) can proliferate and differentiate into vascular endothelial cells (ECs) without expressing phenotype of mature ECs and forming vascular precursors. It includes multiple stages of cells from the mother cells to mature blood vessel ECs. EPCs can promote repairment of ECs, establishment of collateral circulation and restoration of blood supply particularly in ischemic tissues and organs. It can not only involved blood vessel formation in embryonic, but also play an important role in angiogenesis after birth.The main ways of stem cells transplantation are focus on coronary artery injection, endometrial injection, open-chest cardiac injection and autologous transplantation, et al. Studies have shown that adopted those approaches above could improve cardiac function in patients with ischemic heart disease, but those approaches need a high concentration of cells and also involved interventional operation. Transplantation from intravenous injection is a noninvasive way, but a large number of blood cells may migrate to other tissues and organs which may cause unexpected angiogenesis. Therefore, it is necessary to find a way to transplant stem cells by intravenous injection and the cells can shift into targeted tissue.Recent studies have found that as an ultrasound contrast agent, micro- bubbles were not only enhance the gray-scale ultrasound imaging, but also can be used as a new drug or gene delivery vehicles. After ultrasound Irridiation, cavitation and mechanical effects produced by microbubbles destruction could increase permeability of cells membrane, make rupture of microvessel, broaden the endothelial cell gap, so that the target gene could reach the tissue and cells from the broken capillaries and endothelial cell gap, which enhanced the targeted therapy and efficacy of gene. Meanwhile, ultrasound targeted microbubbles distruction (UTMD) can cause local tissue inflammation and accumulation of inflammatory cells, release of inflammatory factors, which can induced some potential biological effects. Researches has shown that ultrasound microbubbles destruction could increase the expression of local vascular cell adhere molecular 1 (VECM- 1), there by, promot MNCs adhere on the endothelium.HIF-1αas an important regulator of oxygen homeostasis transcription factor caused more concern. HIF exist widly in human and mammals, it can promote the expression of more than150 cytokines. It is the most important regulator in the hypoxic response reactions.Based on the theories above, whether Ultrasound microbubbles destruction could be used as a new effective way of stem cells transplantion need to be further explored. Therefore, this subject was to transplant EPCs used ultrasound microbubbles combined with recombinant adenovirus (Ad- EGFP/HIF-1α), then observed the effect of the therapeutic of myocardial infarction, so as to offer a new approach and methods to treat ischemic heart disease.Overall,The study was composed of the following three parts.PARTⅠIsolation, Cultivation and Identification of Endothelial Progenitor Cells from Rat Bone MarrowObjective To investigate the methods of isolating and culturing endothelial progenitor cells (EPCs) from rat bone marrow,as well as their differentiating into endothelial cells. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation, then plated on dishes coated with fibronectin and induced with VEGF, bFGF and EGF for two weeks. The expression of cell markers was assessed by immunocytochemistry, and the attached cells were stained with Dil-ac-LDL and FITC-UEA-1. Results The attached EPCs were able to line up in the typical cord-like structure and formed clusters and cobblestone. Adherent cells showed CD34,CD133,Flk-1 andⅧ/vWF positive in different time and took up Dil-acLDL,bound FITC-UEA-1 double positive fluorescence by laser confocal microscopy. Conclusions The mononuclear cells can be isolated from rat bone marrow using percoll density gradient centrifugation,and after induction using VEGF, bFGF and EGF, we can get high purified EPCs with the same characteristics as the endothelial cells,and EPCs can be differentiated into endothelial cells in vitro.PARTⅡInfection of Recombinant Adenovirus Ad-EGFP/ HIF-1αto Rat Endothelial Progenitor Cells in VitroObjective To investigate the feasibility of infection of recombinant adenovirus Ad-EGFP/HIF-1αto rat EPCs. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocytochemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Expression of EGFP in infected EPCs was observed by fluorescence microscope, cell growth activity was detected by MTT, infection efficiency was detected by flow cytometry, and expression of HIF-1αin EPCs was verified by RT-PCR analysis. Results After EPCs were infected with recombinant adenovirus, green fluorescence was found and increased gradually with the time in EPCs.The survival rates of EPCs had no significant change after infection, and the infection rate increased gradually with the time. HIF-1αwas detected in infected cells by RT-PCR. Conclnsions The recombinant adenovirus Ad-EGFP/HIF-1αcan infect EPCs effectively, without significant effect on activity of EPCs.PART III Combination of Ultrasound Microbubbles and Recombinant Adenovirus Ad-EGFP/ HIF-1αMediate EPCs to Rat Myocadial Infarction SECTIONⅠStudy on Myocardium-Targeted Homing ofIntravenously Implantated EPCs by Ultrasound Microbubbles and Ad-EGFP/ HIF-1αObjective To explore the feasibility of Myocardium-targeted homing of intravenously implantated EPCs by Ultrasound Microbubbles and Ad-EGFP/ HIF-1α. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocytochemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Thrity Sprague Dawley rats were randomly divided into 5 groups after the models of myocardial infarction were prepared.①Blank control group(C),②Ultrasound+Microbubbles group(US+MB),③Endothelial progenitor cells group (EPCs),④Ultra- sound+Endothelial progenitor cells group (US+EPCs) and⑤Ultrasound+ Microbubbles+Endothelial progenitor cells group (US+MB+EPCs). All the rats were sacrificed 48h after EPCs implantation. The intensity of EGFP in the rat myocardium was observed using laser confocal microscopy. The expression of HIF-1αwas respectively detected by Western blot. Results The EGFP intensity in US+MB+EPCs group was the highest among all the groups,and its expression of HIF-1αwas higher than those of other groups. Conclusions Intravenously implantated EPCs by ultrasound microbubbles and Ad-EGFP/ HIF-1αcould augment the homing ability of EPCs to the infracted myocardium, which providing a novel strategy for EPCs implantation of ischemic heart disease.SECTIONⅡCombination of Ultrasound Microbubbles and Recombinant Adenovirus Ad-EGFP/ HIF-1αMediate EPCs to Rat Myocadial Infarction Objective To explore the feasibility of mediated EPCs treating acute myocardial infarction by ultrasound microbubbles and Ad-EGFP/ HIF-1α. Methods The mononuclear cells were isolated from rat bone marrow using percoll density gradient centrifugation and induced with VEGF, bFGF and EGF. The expression of cell markers was assessed by immunocyto- chemistry. Ad-EGFP/HIF-1αwas amplified in HNK293 cells, and then transfected EPCs in vitro. Thrity Sprague-Dawley rats were randomly divided into 5 groups after the models of myocardial infarction were prepared.①Blank control group(C),②Ultrasound+Microbubbles group (US+MB),③Endothelial progenitor cells group (EPCs),④Ultrasound+ Endothelial progenitor cells group (US+EPCs) and⑤Ultrasound+Micro- bubbles+Endothelial progenitor cells group (US+MB+EPCs). All the rats were sacrificed 4w after EPCs implantation. The CD34 expression was detected by IHC, and Microvessel density (MVD) was deternined. The expression of VEGF was respectively detected by Western blot. Results IHC showed that CD of US+MB+EPCs group was the highest among all the groups (84.24±3.80/HP vs 17.50±2.28/HP, 27.69±6.42/HP, 54.42±3.54/HP, 67.80±5.72/HP). The expression of VEGF of US+MB+EPCs group was higher than those of other groups. Conclusions Combination of ultrasound microbubbles and recombinant adenovirus Ad-EGFP/ HIF-1αcould mediate EPCs effectively and noninvasively to the ischemic myocardium, and promote the expression of VEGF and angiogenesis, which providing a novel strategy for gene therapy of ischemic heart disease.