Self-repaired Of Myocardial Ischemia And Necrosis Induced By Isoproterenol In Rats

BackgroundMyocardial infarction is a common cardiovascular disease, therefore in the treatment process of the realization of myocardial regeneration has become the focus and difficult problem. In recent years, some research pointed out that the application of stem cell therapy can be achieved myocardial regeneration and repair. However, the cardiomyocytes can be finish natural restoration is still not confirmed and which cells involved in after myocardial ischemia and necrosis, this experiment study on this issue.ObjectivesThe myocardial ischemia model of rats was set up through the induction of isoproterenol. Using histology and immunefluorescent chemical technology research histopathologic changes in the area of myocardial ischemia and necrotic by using molecular markers Such as c-kit. CD34,Nanog. Gate4, Nkx2.5and gap junctions protein connexin43. troponin CTnI. discusses the biological characteristics of proliferous (migrating) cell in the area of myocardial ischemia and necrotic.Materials and methodsTotally36SD rats (female, male is not restricted) weighting180±20g were provided by the Experiment Animal Center of the Xinxiang Medical College. Animals were randomly divided into the experimental group (1day.4week and8week.8in every group) and control group(8rats), Rats in the experimental group were given intraperitoneal injection of isoproterenol hydrochloride at a dosage of4mg/Kg, continuous intraperitoneal injection for7days; The control group intraperitoneal injection of the same volume of normal saline. Animals in each group using limb lead ECG assessment the model is successful or not. After injection, respectively,on the1st day,4th weeks and8th weeks, rats were anaesthetized with10%chloral hydrate solution. Immediately opened their thoraces and took out hearts, Both the atrial tissues and the right ventricular free wall were cut carefully, then retained about1/2of the left ventricular inferior wall tissues. Each group rats were rapidly taken their tissues in the subendocardial myocardium as materials of electron microscope, Quickly fixed in2.5%glutaldehyde solution, post fixed in1%osmium tetroxied, routine steps of desiccation and clearing, embedded in Epon812preparing for ultrathin sections, stained with uranyl acetate and lead nitrate.and then observed under H-7500electron microscope.Other cardiac wall tissues fixed in4%paraformaldehyde. By20%,30%sucrose gradient, embedded in the OCT, sliced on a freezing microtome, kept in the-20°refrigerator prepared for using. Detection:①HE staining was used to observe morphology more clearly under the light microscope level in4groups of rats;②the structures of myocardial ischemia and necrosis tissues were observed by electron microscope;③The immunofluorescent technique was used to detect the expression of differentiation of molecular markers in the ischemic and necrosis areas, such as stem cell molecular markers (CD34, c-kit. Nanog). cardiac transcription factors(Nkx2.5and GATA-4) and myocardial cell markers (Connexin43and CTnI). and the experiment divided into negative control groups, in which the blocking buffer was used to replace the first antibody, others remained the same steps. The fluorescent images were observed and acquired by Olympus FV-1000laser scanning confocal microscopy.Result①After myocardial ischemia was induced by isoprenaline hydrochloride, the limb lead ECG appeared typical elevated ST-segment in rats.②The HE staining showed that the ischemia and necrosis area in the subendocardial myocardium present weak staining, and mainly focused on the left ventricular wall and the interventricular septum which near the endocardium and the inner layer of myocardium, especially the cardiac apex. ③Myocardial ischemia and necrosis tissues were observed by electron microscope, in those areas also appeared fibroblast proliferation, Interstitial deposition of fibrous matrix, the formation of new blood capillary and cells proliferation.④In the ischemic and necrosis zone, the surface markers expression of stem cell such as Nanog appeared in the early stage, CD34and cardiac transcription factors Gate4appeared in the middle stage, Connexin43and troponin CTnI appeared in the later stage. The positive results of CD34are mainly localized in the cytoplasm, linear distribution and staining clear; The positive results of Nanog are mainly localized in the cell membrane, cord distribution and staining clear; The expression of Gate4occurred translocation in different stages,early localized in the nucleus, later transferred to the peripheral area of the nuclear and staining clear; The expression of Connexin43localized in the cytoplasm and cell membrane, interspersed and shaped distribution, staining clear; The expression of CTnI localized in the cytoplasm, interspersed and shaped distribution, staining clear.ConclusionWithout any special interfering conditions, the natural restoration of cardiac was affirmed and proved that there exist some stem cells that can attend the self-repairing of myocardial tissue in the myocardial ischemia and necrosis area, forming some "cardiac-like cells"which have the characteristic of myocardial cells.