Experimental Research On Treatment Of Acute Myocardial Infarction Induced By Hepatocyte Growth Factor Mediated By Ultrasound-Targeted Microbubble Destruction In Rats

Coronary Heart Disease is a serious threat to life and health of human being. Although coronary angioplasty and coronary artery bypass surgery have saved the lives of many patients, the condition of some serious patients has not been significant improvement. Gene therapy for the treatment of these patients with serious coronary artery disease can provide a new treatment strategy, since it still lacked a safe, stable and efficient gene transfection method which can limit gene therapy in a wide range of clinical applications. Recent studies have showed that ultrasound-targeted microbubble destruction (UTMD) is a new noninvasive gene transfer technology, which can provide a new method of gene therapy on coronary heart disease.This study had constructed a eucaryotic co-expression vector (pIRES2-EGFP/HGF) with EGFP and HGF,and prepared a ultrasound contrast agent which can load with gene efficiently. The UTMD was applied in treatment of AMI induced by HGF in rats, then the expression of HGF and EGFP on infarcted myocardium were detected. The study would provide reliable experimental evidences for studying and evaluating gene transfer mediated by UTMD, which could be a novel method in gene therapy of CHD. The study included the following three parts:PARTⅠ: Construction and expression of enhanced green fluorescent protein and HGF co-expression vectorObjective To construct EGFP and HGF co-expression vector and to detect its expression in cultured eukaryocyte. Methods A pair of primers of HGF with SacⅠand BamHⅠwere designed and constructed, then PCR was performed. The PCR products were inserted into cloning vector pMD18-T to construct the recombinant plasmid of pMD18/HGF,and the recombinant were transferred into JM109.The PCR of colony, restriction analysis, and DNA sequencing were performed to identify the correctness of the recombinant .The HGF gene fragment obtained from pMD18-T/HGF that was digested with SacⅠand BamHⅠ,and then inserted into pIRES2-EGFP that was cut with SacⅠand BamHⅠ. The recombinant pIRES2-EGFP/HGF was identified with restriction analysis. The expression plasmid pIRES2-EGFP/HGF was transfected into HUVEC mediated by liposome reagent, then the expression of EFGP in cell were observed by fluorescence microscopy and HGF protein was detected with Western blot. Results The sequence of the cloned DNA fragment was identical to HGF that was reported on Gene bank, and the HGF gene was inserted into eukaryotic expression vector pIRES2-EGFP correctly. The recombinant expression plasmid was successfully transferred into HUVEC observed by fluorescent microscope and effective expression of HGF was also testified by Western blot. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HGF was successfully constructed and effectively expressed in HUVEC.PARTⅡ: Experimental study on preparation and characteristics of lipid microbubbles loading geneObjective To prepare self-made lipid microbubbles loading gene, to study its characteristics and the gene carrying ability in vitro, and to assess its enhancement effects in contrast imaging on normal rabbit kidneys. Methods The Lipid microbubbles were prepared, and its appearance, concentration, mean diameter and potential were detected before and after irradiance of 60CoγRay, and the gene loading ability of the microbubbles were detected. Seven normal rabbits were examined to observe the enhancement time and intensity in kidney parenchyma. Results The mean diameter range of the self-made lipid microbubble was 2.11-6.43μm, the mean diameter was 2.79μm and the concentration was (3.16±0.29)×109 /ml.Those indices were not changed significantly after sterilization with irradiance of 60CoγRay. The maximum ratio and amount of loading gene of microbubbles was 22 % and 0.45 mg/ml respectively. The appearance and diameter of microbubbles did not changed obviously after gene loading. Contrast imaging showed that the self-made lipid microbubbles could enhance the intensity of gray-scale (GS). Conclusions The self-made lipid microbubbles are consistent to standard of ideal ultrasound contrast agent, which may become a new ultrasound contrast agent and a vehicle for encapsulating with genes or drugs.PARTⅢ: Experimental research on therapeutic angiogenesis induced by HGF directed by ultrasound-targeted microbubble destructionObjective To explore the feasibility of therapeutic angiogenesis in myocardial infarction induced by HGF mediated by ultrasound-targeted micrbubble destruction, and to evaluate its therapeutic efficacy. Methods Forty Wistar rats were divided to 4 groups after the models of myocardial infarction were prepared, such as①HGF+ultrasound+microbubble groups(HGF+US/MB),②HGF and ultrasound group(HGF+US),③HGF and microbubble group,HGF+MB) and④surgery alone group(SA). Ultrasound-targeted destruction microbubble loaded with HGF gene with ECG trigger was performed in HGF+US/MB group , and HGF gene with ECG trigger in HGF+US group.Microbubble loaded with HGF gene were infused intravenously in HGF+MB group, and normal saline were infused in surgery alone group. All the rats were killed after transfected 14 days. The EGFP expression was examined in tissue of myocardium, liver and kidney in all groups by fluorescent microscope, the CD34 expression were detected by IHC, and MVD were counted in high power field. The HGF expression on myocardium was detected by Western blot and ELISA, and the correlation between the contents of HGF and MVD in myocardium was analyzed. Results The EGFP expressions were detected on myocardium of HGF+US/MB group, and the expressions on anterior wall were more than those of posterior wall, but no EGFP expression was detected on liver and kidney of all groups. A few expressions of HGF were detected only on small vessels and capillary endothelium, and no expression in SA and HGF+MB group. IHC results showed that CD34 expressions were shown as brown granules, which located on membrane and endochylema of vascular endothelial cell. The results of MVD by microscope showed that the MVD of HGF+US/MB group were the highest among all the groups(266.9±39.83/HPF). The results of Western blot showed there existed a protein strap (69 kD) in HGF+US/MB group, and an weak protein straps in HGF+US group. the results of ELISA showed that the contents of HGF in myocardium were the highest in HGF+US/MB group(5.54±0.81 ng/g), and the contents of HGF in anterior wall were more than those of posterior wall(P<0.05). There were significant difference between HGF+US/MB group and others groups (P<0.01). The correlation analysis results showed there existed a positive correlation between the contents of HGF and MVD in myocardium. Conclusion Ultrasound-targeted microbubble destruction could deliver HGF into infracted myocardium and produce angiogenesis effect, which could provide a novel strategy for gene therapy of myocardial infarction...