Treatment Of Acute Myocardial Infarction In Rats By Ltrasound Microbubble Mediated ShRNA-PHD2 Transfection

[Objective] HIF-1? is a key regulator of PHD2 degradation. When myocardial ischemia and reperfusion, up regulation of HIF-1? is able to activate the downstream angiogenesis gene. Although the application of naked plasmid or viral vector gene transfer in the treatment of cardiovascular diseases has been a success, but gene therapy still exist many unsolved problems, such as the lack of safe and effective transfection system, lack of stable gene expression and transcription of the host immune response. Ultrasound targeted microbubble destruction technology as a non-invasive, new and efficient gene transfer method for the treatment of ischemic heart disease gene therapy to provide a new way of gene transfer and treatment. In this paper, the aim of this study is to study the treatment of acute myocardial infarction in rats by PHD2 sh RNA mediated by ultrasound microbubble targeted disruption.[Methods] Acute myocardial infarction model in Sprague-Dawley rats was established. First, thirty rats were divided into plasmid, Plasmid+US and Plasmid +UTMD three groups. Rats in 4 days after transfection were acquiring cardiac tissue frozen sections were made and fluorescence microscope to observe the EGFP protein expression in the infarcted myocardial tissues of rats in each group. Then, the 60 Sprague-Dawley rats were randomly divided into three groups: sham group, MI-EGFP group and MI-sh PHD2-EGFP group. Then EGFP-MI, MI EGFP-sh PHD2 rats respectively via the tail vein injection MB /PEI/EGFP complexes, MB /PEI/ sh PHD2 EGFP mixture; at the same time MI-EGFP group, MI-sh PHD2- EGFP group rats using ultrasound gene transfection apparatus in the treatment of irradiation in the area before the heart, and irradiation conditions as: 2.0W/cm2, 3min, 20%DC, IMHZ. Rats in group Sham were injected with the same amount of normal saline by tail vein. Rats at 4 weeks after transfection were then performed RT-PCR and Western blot were used to detect the expression of PHD2, HIF-1a and VEGF m RNA and protein; immunohistochemistry(IHC) staining method was used to detect the myocardium in the infarct border zone of HIF-1?, VEGF and microvessel density(MVD). Masson staining to detect myocardial infarction area percentage. The left ventricular systolic function was detected by echocardiography in the rats 4 weeks after ultrasound therapy.[Results] 1, laser confocal microscopy showed that under High magnification,plasmid group, see a few positive cells expression, Plasmid+US see the number of positive cells and Plasmid+UTMD group multiple positive cells expression; compared with group Plasmid+US, Plasmid+UTMD group gene transfection efficiency is higher, difference has statistical significance(P < 0.05). 2, RT-PCR showed the lowest expression of m RNA MI-sh PHD2-EGFP in the PHD2 group, while the expression of HIF-1? and the downstream Proangiogenic factor m RNA were the highest, the difference was statistically significant(P<0.05). Western blot showed the group PHD2 protein MI-sh PHD2-EGFP expression was the lowest, and HIF-1? and downstream Proangiogenic factor protein was the highest, and the difference was statistically significant(P < 0.05). 3, MI-EGFP compared, the immunohistochemical staining showed that the MI-sh PHD2-EGFP group for HIF-1?, VEGF expression were increased, the difference has statistical significance(P < 0.05). 4, the comparison with MI-EGFP, MI-sh PHD2-EGFP treatment group, the microvessel density increased, the difference was statistically significant(P<0.05). 5, after myocardial infarction, left ventricular become enlargement, ventricular wall become thinning, compared with the MI-EGFP group, MI-sh PHD2-EGFP group, the proportion of wall thinning is less; while compared with the MI-EGFP group, the MI-sh PHD2-EGFP group is relatively small secretion of collagen;6, the preoperative heart function in each group no significant difference. In postoperative 4 weeks and group MI-EGFP comparison, MI-sh PHD2-EGFP group LVEF, LVFS were increased, the difference has statistical significance(P < 0.05).[Conclusion]Ultrasound targeted microbubble significantly increased sh PHD2-EGFP transfected into rat ischemic myocardial tissue and PHD2-sh RNA can inhibit the expression of PHD2 gene, promotes HIF-1? and downstream Proangiogenic factor gene expression, form more neovascularization, effectively improve heart function and prevent myocardial remodeling.