The Efficacy Of MSCs Implantation By Diagnostic Ultrasound Mediated Microbubbles Destruction On The Improvement Of Cardiac Function At The Late Time Of Rats Myocardial Infarction

Coronary heart disease(CHD)is one of the most common disease,it will lead the heart fuction to failure which harm people's health severity.Myocardial ischemia(MI)associated with coronary artery disease is a leading cause of morbidity and mortality in clinical practice.Reeently,beeause of the treatment methods(ineluding iniervention?Peration and drug treatment),The infarct-relatedartery can be reperfused effeetively.But MI has usually generated quiekly before the opening of the infaretion artery.So the infarction of the myoeardial function can not be improved by these treatments.Myocardial infracted area was gradually replaced by the fibrous scar tissue,and eventually it will evolved into irreversible heart failure.Transplantation of stem cell is a promising method for the treatment of MI.Several studies have indicated that bone marrow derived mesenchymal stem cell(MSCs)offer a novel therapeutic option in the treatment of heart diseases.MSCs can secrete vascular growth factors,enhance angiogenesis,improve heart function and differentiate into endothelial cells or cardiomyocytes in a specific environment.The best transplantation time of MSCs in the treatment of MI was in the early stages of MI(3?7days after MI).But the pro-homing factor expression levels of myocardial microenvironment were significantly reduced in the late stage of MI.Fibrous scar tissue has formed at this time.The effection of MSCs transplantation was poor or invalid.Diagnostic ultrasound target microbubble destruction(DUTMD)has been found to produce micro vascular permeabilization?microvascular ruptures and endothelium interspace widen,it also stimulate the secretion of endogenous vascular growth factors and promote angiogenesis.The microenvironment will be changed and inflammatory response will be induced after DUTMD.Mild inflammatory response may be helpful for the mobilization and homing of stem cells.First the expression of cytokine and adhesion molecules in the infarcted myocardium induced by various mechanical index of DUTMD will be investigated,then the proper ultrasound intensity which is suitable for MSCs transplantation will be determined in this study.MSCs transplantation will be performed after myocardial environment changes induced by DUTMD and the effection will be evaluated.Part I:Isolation,cultivation and identification of rat bone marrow-derived mesenchymal stem cells in vitroObjective To investigate a stable method for the isolation,purification,cultivation and identification of bone marrow-derived mesenchymal stem cells in vitro.Methods Rat bone marrow mesenchymal stem cells(MSCs)were separated by adherent cell separation?The MSCs were cultured in DMED/F12 with 10%fetal bovine serum and MSCs were purified by adherence to the wall of flask.When cells become more than 80%confluence,the adherent cells were digested using 0.25%trypsin and began to sub culture and proliferation.The 3rd to 5th passage of MSCs were chosen as all experimendtal use.MSCs were trypsinized and characterized by flow cytometry using antibodies against cell surface markers CD45,CD34,CD166,CD44,CD90,and CD29.MSCs were induced to differentiate into fat cells and osteoblasts cells.Results Most of cultured MSCs were spindle shaped and became more uniform after several passages.MSCs were able to proliferate actively and grew faster after reseeding.The growing peak was reached at the 7 days.Our results revealed that MSCs expressed the positive for CD45 and CD34,but the negative forCD166,CD44,CD90 and CD29.A multilineage differentiation was successfully induced that the cells could differentiate into adipocytes and osteocytes.Conclusion Bone marrow mononuclear cells were separated by adherent cell separation.High purity MSCs can be obtained by passaged MSCs,which may provide sufficient cell sources for rat cadiomyocytes replacement therapy.Part ?:Establishment and evaluation of rat myocardial infarction modelObjective The model of rats myocardial infarction was successfully established.Methods Male Wistar Rats weighing 220g to 250g were anesthetized using 3%pentobarbital sodium(40mg/kg)via intraperitoneal injection.Rats were intubated and ventilated with a rodent ventilator.Proper intubation was confirmed by observation of the chest expansion and retraction during ventilated breaths.During the operation,the rats were kept warm.The chest was opened between the fourth and fifth ribs,the pericardium was opened,and the anterior descending branch of the left coronary artery(LAD)was ligated with a 5-0 silk suture.For the ischemia-reperfusion experiments,the ligature was removed to restore perfusion after occlusion for 45min.The blood flow was confirmed by visualization of a return of red color to the previously pale region of myocardium.Then,the chest was closed in layers with a 5-0 silk suture.Results After MI model were builded by ligation of LAD,LV systolic function was attenuated greatly by M-mode ultrasound,a blue dye was detected in the MI area with Masson staining.Conclusion A stable rat MI model can be successfully established by the left anterior descending coronary artery ligation method.Part III:Effects on myocardial microenviroment by diagnostic ultrasound-mediated microbubbles destruction in a late-time myocardial infarction modelObjective To explore the effects on myocardial microenviroment induce by diagnostic ultrasound with different mechanical index and to determine the proper ultrasound intensity which is suitable for MSCs transplantation.Methods The eligible MI rats were randomly divided into five groups after 14 days after MI,rats in group 5(n=3)were irradiated with a diagnostic US system at a 2 s trigger time,with a mechanical index of 1.9,focused on the anterior left ventricular wall,and continued irradiated for 10min,a long with the intravenous injection of 1ml microbubbles(MB).Rats in group 2(mechanical index of 1.6 US+MB,n=3),group 3(mechanical index of 1.0 US+MB,n=3),group 2(mechanical index of 0.6 US+MB,n=3)and group 1 mechanical index of 0.4 US+MB,n=3)received UTMD with a mechanical index of 1.6,1.0,0.6 and 0.4 in the same way as group 1,respectively.control group(n=3)received no UTMD.After lday after UTMD,the rats were sacrificed and the hearts were harvested.The expression of VCAM-1,SDF-1,and VEGF mRNA and protein were detected by Western blotting.Results The results from western blot revealed that mechanical index= 1.0?1.6?1.9groups markedly increased the protein expression of SDF-1?VCAM-1?VEGF compared with the mechanical index =0.4?0.6groups and non-treated control group(P<0.05,and 2?3 fold).There was no significant changes with each others of mechanical index =1.0?1.6?1.9groups.Conclusion Diagnostic ultrasound-mediated microbubbles destruction of mechanical index=1.0?1.6 and 1.9 can significantly improve the microenvironment resulting in the beneficial of the homing and differentiation.Part IV:Study the efficacy of MSCs implantation by diagnostic ultrasound-mediated microbubbles destruction on the improvement of cardiac function at the late-time of rats myocardial infarctionObjective To study the efficacy of intravenous MSCs implantation by diagnostic ultrasound-mediated microbubbles destruction on the improvement of cardiac function after the late time of rats myocardial infarction.Methods(1)MSCs were labeled by DAPI.After 72 hours of cell transplantation,the survival of implanted cells was identified by the number of DAPI-positive cells in frozen sections made from of MI under fluorescent microscope in the DAPI-MSCs group and diagnostic US+microbubbles+ DAPI-MSCs group.(2)After 4 weeks of MSCs treatment,echocardiography was performed to assess the cardiac function,myocardial collagen fiber area was analyzed with Q-Win images software with Masson staining to give evidence of inhibition in fibrosis by this implantion method.Results The DAPI-positive cells were located in the infarcted and border area,while there were nearlt none in the normal myocardium.Quantification analysis of DAPI-posive cells in the infarcted and border area showed that the number of DAPI-positive cells in the US+MB+MSC group(133±38)was much more than that of the MSCs infusion group(55±24)at 200 magnification field under fluorescent microscope(p<0.01).(2)Indeed,4weeks after transplantation,FS%was 23.40±4.15%in PBS group,22.67±2.82%in US+MB group,27.77±3.31%in MSC group and 32.05±3.85%in US+MB+MSC group(p<0.05 in US+MB+MSC and MSC groups vs.saline and US+MB groups;p<0.05 in US+MB+MSC group vs.MSC group).(3)The collagen percentage area(%)of US+MB+MSC group was significantly decreased when compared with US+MB and PBS groups(all p<0.05).Conclusion In the late time of myocardial infarction,therapeutic effects will be maekedly enhanced by MSCs transplantation after the myocardial microenvironment changes induced by diagnostic ultrasound targeted microbubble destruction,which provide a new chance for MSCs transplantation again.