| BackgroundIn modern society, the incidence of ischemic heart disease is increasing year by year. The main therapeutic method include promoting and restoring coronary blood supply, reducing myocardial ischemia, and attenuing coronary arteriosclerosis. CABG and PCI is major treatment. Despite the rapid progress of treatment, there are some patinets with chronic total or diffused coronary occlusion are not suitable for PCI or CABG, besides that some patients with recurrence after operation can not tolerate these kind of revascularization operation again. In response to these clinical problems, how to save myocardium and improve cardiac function for these kind of patients has become a problem that plagued the medical profession. The recent proposal of "therapeutic angiogenesis" concept for such patients has brought new hope, which include "paving the way cells", "Molecular bypass" and "gene therapy". While a large number of basic research confirmed that these three methods can promote angiogenesis in ischemic tissues, but the results of clinical trials are still controversial.Therefore seeking an effective method of promoting angiogenesis is particularly important. Low frequency pulsed magnetic field(LFPMFs) has been shown to have effect of anti-inflammatory, analgesic, lipid-lowering, improving microcirculation and modulating the immune activity and promoting bone healing, can also affect the behavior of animals and humans, and there are the promotion of human umbilical vein endothelial cells and bovine aortic endothelial cell proliferation, migration and other biological effects, moreover it's no invasive, safe. So it prompted LFPMFs may be cost-effective method of promoting angiogenesis, but there is no study and report about LFPMFs on ischemic heart,In this study, we will take myocardial infarction model in rats and cardiac microvascular endothelial cells, observe and investigate the low frequency pulsed magnetic field on the treatment of ischemic myocardial angiogenesis and its possible mechanism.Objective1. Develope a pulsed magnetic field treatment, setting the magnetic field strength and frequency can be adjusted within a certain range, and prepare a induction coil. for cell and animal studies.2. Establishe myocardial infarction model in rat, identify whether pulsed magnetic field can promote angiogenesis in ischemic myocardium or not and effective magnetic parameters, and elucidate the possible mechanism.3. Purify and culture cardiac microvascular endothelial cells, identify the effect of low frequency pulsed magnetic field on proliferation and migration of cardiac microvascular endothelial cell. Methods1. According to electromagnetic theory,designed and developed a pulsed magnetic field treatment instrument, pulsed magnetic field generated by the microcontroller, high-speed measurement of induction coil current sensor for the automatic pulse current intensity, the peak holding circuit through the magnetic field strength measurements is transported to the monitor, the pulse magnetic field to drive both the multi- mode output. Magnetic field frequency and field strength can be adjusted, we made a stent with plexiglass in the area of effective uniform magnetic field. In this study we take pulse square wave.2. For MI model, we used 180 ~ 200g selected male 8 weeks old SD rats, exposed heart through the left 3-4 intercostal space, and ligated the left anterior descending of coronary artery.3. Catheterization for cardiac function and eight polygraph recorder for electrocardiogram, arterial blood pressure,±LVdp / dtmax and other physiological indicators.4. Immunohistochemical method for capillaries in border, them randomly selected areas under a microscope and analyzed capillary density with software; and for detecting expression of specific proteins of vWF and VIII in cardiac microvascular endothelial cells.5. TTC staining for infarcted area.6. ELSA Kit for concentation of VEGF,bFGF in body circultion.7. Protein mass spectrometry for detecting different proteins in ischemic myocardium8. HE staining for morphological structure of important organs. 9. Sterile isolated adult rat left ventricular tissue, removed those internal and external membrane and coronary artery tissue, myocardial tissue were cut into 1mm3 by enzymatic digestion, cultured the cells in special mediam.10. Test of uptaking Dil-Ac-LDL for indentifing CMECs11. Flow cytometry(FCM) for measuring the purity of CMECs12. Western blot for determining protein concentration of VEGF,KDR(VEGFR2),bFGF,FGFR2 in borded.13. MTT test for proliferation of CMECs.14. Scratch assay for CMECs average migrative distance.15. Millicell for Mobility of CMECs.16. Nitrate reductase kit for NO release of CMECs.Result1. Myocardial infarction by the 15Hz 6mT field 2 hours a day for 2 weeks, can significantly improve heart function, reduced myocardial infarct size, and to promote angiogenesis in ischemic infarct border zone.2. Biological effects of 15Hz 6mT magnetic fields may be related to the increasing of VEGF, KDR (VEGFR2), bFGF, FGFR2 protein expression in ischemic myocardiu, and promoting the level of VEGF, bFGF concentration in circulation blood.3. 15Hz 6mT magnetic field can promote cardiac microvascular endothelial cells proliferation, migration and secretion of NO... |
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