| Backgrounds and aimsBecause of increased survival of patients and in age of death in the population, half the deaths in the world are caused by coronary atherosclerotic heart disease. Myocardial infarction is a leading cause of morbidity and mortality in human . The disease will be the biggest cause of death worldwide in the near future. There is increasing evidence that stem cell mobilisation to the heart and differentiation into cardiac myocytes is a naturally occurring process. However, it occurs too slowly for there to be meaningful recovery of left ventricular function after myocardial infarction. The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in loss of cardiac function, ventricular remodeling, and progressive dysfunction leading to congestive heart failure. Current therapies address this process at two main points: (1) Reducing the duration of ischemia by surgically, percutaneous transluminal angioplasty (PTCA, pharmacologically removing the vascular blockage; but there are increasing numbers of patient with extensive atherosclerotic coronary artery disease not amenable to traditional methods of revascularization). (2)By using drug therapy (ACE-inhibitors and beta blockers) to mitigate ventricular remodeling. While these approaches have had a substantial impact in the management of heartattacks there remains a considerable amount of room for improvement. The ideal therapy would have the following activities; it would minimize loss of cardiomyocytes by reducing cell death, promote return of stunned and hibernating myocardium to normal function, stimulate revascularization of the ischemic region by enhancing angiogenesis, and regenerate viable cardiomyocytes to replace those lost to the initial ischemia thereby preserving contractile function and reducing the opportunity for scarring. Recent advances in biotechnology and in the understanding of tissue regeneration have allowed development of novel therapeutics with the potential to approximate this ideal. One novel way to treat this process after myocardial infarction is cell therapy. Cellular transplantation has focused on use of various cell types, including differentiated cells such as skeletal myoblasts, cardiac myocytes, and bone-marrow -derived cells. Different methods have been used to repair infarcted myocardium after acute myocardial infarction. Of these, the most important is "biointerventional therapy," which mainly involves stem cells derived from bone marrow. Bone marrow contains multipotent adult stem cells, which have a great capacity of directionally differentiating to myocardium. Although many cell types related to bone marrow contribute to organ repair in infarction models, bone marrow mesenchymal stem cells (BMSCs) have the greatest potential for repairing myocardia. BMSCs have long been considered "second-class citizens." Recently, it became clear that a small population of BMSCs in the bone marrow includes putative adult stem cells with important functional features, such as the potential for rapid self-renewal, giving rise to a wide variety of connective tissues, includeing bone, cartelage, muscle, fat, and perhaps neuronal cells. BMSCs have two advantage ,first it is convenient to collect and the process do little harm to body , second ,it are restricted by MHC(major histocompatibility complex). The purpose of the present study was as follows (l)The feasibity of culture and expansion of BMSCs in vitro.(2)Whether transplanted BMSCs by intravenous could reach in the infracted myocardium.(3)Whether in scar transplanted BMSCs could transdifferentiate into cardiomyocytes in the appropriate Niche and express cardiac specific mak ersarcomeric actin.(4)To assess the functional improvement in rat left ventricular with coronary ligation after transplantation by intranvenous.Materials and methodsIsolate and culture of BMSCs :130- 150g SD rats,under general anesthesia, the fermoral and tibial bone marrow specimens were extrarcted by steritle syringe containing 10% fetal bovine serum and DMEM cell suspendsion were centrifuged at lOOOrpm for 10 min .Then discarded upper clear liquid . Cells isolated from bone marrow in 5ml DMEM medium were then introduced into plastic culture bottle and incubated with 95% air and 5% CO2 at 37°C.The medium was completely changed 24 hours later in order to discard nonadherent cells and subsequently replaced every 3 days.When cell reached about 90%,the adherent cells were released from the culture with 0.25% trypsin and replanted to two bottle ,after passaged 4 cell became pure sterile bromodexoyuridine solution was added to the culture medium at a final concentration of brdu of 5u g/ml culture medium for 48 hours immediately before transplantation.Creation of model: Briefly, after anesthesia with intraperitoneal injection of 30 mg/kg sodium pentobarbital, the rats were intubated and ventilated via a rodent respirator. Parasternal thoractomy between the 3rd and 4th ribs was performed. The proximal left anterior descending branch (LAD) of coronary artery was ligated using 5-0 slik sutures in MI rats. The thoracotomuscle and skin layers were closed with 3-0 slik sutures. Divided group and cell transplantation :50 male SD rats were divided randomly into four groups: A :acute myocardial infarction + anti-inflammation +rMSC intravenous transplantation group (n= 15). B :acute myocardial infarction + rMSC transplantation group (n = 15) .C: acute myocardial infarction +anti-inflammation group (n= 10). D: acute myocardial infarction control group (n = 10). Hmodynamics and analysis echocardiographic studies: After anesthesia with intraperitneal injection of 0.2g/kg chloral hydrate, Two-dimensional echocardiography was performed with a phased-array echocardiographic machine, using A 12MHz pediatric tranducer obtained after surgery . Long-axis, short-axis, andsubcostal views were obtained in bidimensional echocardiography. Regional fractional shortening was calculated by dividing the end-disatolic dimension minus the end- systolin dimension by the end diastolic dimension times 100, expressed as a percentage .Rats were prepared for hemodynamics analysis. Midline incision of neck was adopted to expose right common carotid artery. About 1.0 cm in length of the artery was dissociated for catheter intubation. Left ventricular catheter filled with heparin solution was inserted into left ventricle through the artery. Left femoral artery was prepared for another catheter intubation. The pressure data gathered from artery and left ventricle together with electrocardiogram were analyzed by a computer based biosignal analysis system. Immunohistochemistry After finishing the hemodyn amic studies, the rat hearts were arrested in diastole by intravenous injection of 10% KCL. The heart was removed and then divided transversely at the level of the papillary muscle. The distal portion was collected for HE and immunohistochemical staining studies. Myocardium specimens embedded with paraffin were serial sectioned to a thickness of 3^m, dewaxed and rehydrated. Statistical analysis. All data were given as mean±SD. The comparisons between >2 groups were made using multivariate ANOVA of independent groups to determine the overall difference, and SAK test was used to determine statistical signifi cance between groups. Probability values <0.05 were considered statistically significant.ResultsEchocardiographic :There was significant improvement in cardiac function in A and B group ,as evidenced by decrease LV end-diastolic diameter (LVEDd, P<0.05), increase ejection fraction , compared to C and D group (EF, P<0.05) and fractional shortening (FS, /><0.05).The treatment of stem cells could markedly attenuate LV dilatation and ameliorate LV function(P<0.05). Hemodynamics analysis In C and D group, LV end diastolic pressure (LVEDP) significantly increased (P<0.05), while LV systolic pressure (LVSP) and LV pressure maximal rate of rise and fall (+/-dp/dtmax) significantly decreased (P<0.05). Compared to D group, LVEDP significantlydecreased in A and B group (P<0.01), while +/-dp/dtmax significantly increased (P<0.05).there is a difference in A and B(P<0.05). No lymphocyte proliferation and immonologic rejection were seen in the cardiac tissues of the rats implanted with rMSC. Brdu-labeled rMSC with bule nuclei were distributed extensively in the myocardium of the rmsc tranlpantation groups, ovoid in shape and arranged in parallel with the cardiac muscle fibers,.Little bule nucleus was seen in the normal cardiac tissues. Troponin were positive immunohistochemically in the implanted rMSC with bule nuclei and yellow cytoplasm in A and B, however, negative in the implanted rMSC with bule nuclei and normal cardiac muscle in C and D . Cardiac function has been obviously improved in A and B .There was no significant difference respectively between C and D.Conclusions(1) the rat bone marrow mesenchymal stem cells are suitable to isolate ,culture ,epand and transplant (2) the transplanted BMSCs can reache in myocardial scar after intravenous inject. (3)the transplanted BMSCs expressed cardiac specifc protein and transdifferentiated into cardiomyocytes-like phenotyle in the infarct region.(4) intranvenous and anti-inflammmation can improve rat cardiac function.
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