| Myocardial infarction (MI), leading to irreversible loss of cardiomyocytes and scar formation, is the leading cause of congestive heart failure. The identification of stem cells capable of contributing to tissue regeneration has raised the possibility that cell therapy could be employed for repair of damaged myocardium, and bone marrow-derived mesenchymal stem cells (MSCs) is considered to be an effective therapeutic approach for MI both in basic researches and clinical trials.Mechanisms underlying may involve direct regeneration of the lost cardiomyocytes, and other cell types constituting the cardiac tissue .But there is much debate over the frequency of this phenomenon. The low periimplantation viability of the injected cells may limit the capacity of this mechanism to result in meaningful cardiac regeneration.Researchers have done to enhanced the cardioprotective effects of MSCs by genetic modification, however, the safety and feasibility of genetic therapy is still controversial.Hypoxia has previously been shown to have a critical role in the development of placenta, early human embryogenesis takes place in an environment with a very low oxygen tension. Bone marrow is also a hypoxic place of residence of MSCs. In particular, culturing MSCs at reduced oxygen tension (5%) increased their bone-forming potential, stimulating MSCs with anoxia increased their paracrine effect.We , therefore, hypothesized that MSCs protected ischemic myocardium through paracrine effects that could be further augmented with preconditioning, we stimulated MSCs with anoxia before transplantation ,studied its potential mechanism in protecting the infracted heart. Material and methodsMSCs were acquired from the bone marrow of rats.They were isolated,purified and cultured .Primary culture of neonatal SD rat CMs was prepared as previously described.After 10 days' incubation, CMs grew to about 90% confluence and entered into the experiments..In vitro, CMs were randomly divided into four groups: DMEM, MSCs medium Group , AP -MSCs medium group and control group. Each was treated with anoxia: the CMs were incubated at 37 °C in the anoxic chamber for 24 h. Control group was incubated in DMEM, 20% fetal calf serum under standard cell culture conditions .Apoptosis of CMs was determined by Annexin V-FITC Apoptosis Detection Kit (BioVision, USA)according to the manufacturer's instruction. Cells were visualized directly on glass slides under fluorescence microscopy and analyzed by flow cytometry.In vivo ,Fifty-two Sprague-Dawley male rats (250±30g, body weight)were divided into four groupe, DMEM(G-1), AP-MSCs medium(G-2), MSCs (G-3)and AP- MSCs(G-4).Thirteen animals were used in each groupe.Rats were undergone ischemic procession by thoracotomy and ligation of the left anterior descending coronary artery(LAD) . left ventricles were randomly injected with the following: inspissate DMEM(G-1), inspissate AP-MSCs medium(G-2), MSCs (G-3) and AP-MSCs(G-4) (5 ×10~6cells in total which suspension with inspissate DMEM,150μl) at one week after coronary ligation in rats.After one week, the animals were euthanized and the hearts removed.Five animals in each group. The excised heart was cut into 3 transverse sections and embedded in paraffin. Sections (5-um thick) were cut and mounted on adhered slides. Apoptotic cardiomyocytes in MI were evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay .Apoptosis associated pretein BCL-2, BAX through Western blotting. After four weeks Cardiac function was assessed by transthoracic echocardiography. After echo-cardiographic measurements, the excised heart was cut into sections (5-μm thick). The section was stained with hematoxylin and eosin for arteriole density, Masson's trichrome for remodeling. ResultsIn vitro: Normal cultured CMs showed a low apoptotic rate, the rate increased after anoxia, and could be alleviated by treating with AP-MSCs medium. In vivo:1,After transplantation, left ventricular diastolic dimension (LVDd) and left ventricular systolic dimension (LVDs) were significantly decreased( G-1:0.75±0.06 vs G-2: 0.63±0.02; G-3:0.57±0.02; G-4:0.49±0.03,p<0.01 ; G-1:0.60±0.05 vs G-2: 0.40±0.02; G-3:0.36±0.02; G-4:0.28±0.02,p<0.05).Fractional shortening (FS) were significantly increased; AP-MSCs was predominant after transplantation (G-1:20.94±1.20 vsG-2: 36.4±2.39 G-3:37.12±2.00 G-4:43.32±1.82, p<0.05; G-4 vs G-2,p<0.05)o2, TUNEL labeling was performed at 1 weeks after transplantation in the four groups. The results showed that the number of TUNEL-positive cells was significantly reduced ( G-1:15.09±1.76 vs G-2: 8.97±3.21; G-3:11.77±4.05; G-4:7.93±1.47,p<0.05), and AP-MSCs had the predominant effect. ( G-4 vs G-3, p<0.05)AP-MSCs increased the expression of Bcl-2.3 , The numbers of blood vessels were increased at the boundary of infarction site in the animals transplanted with AP-MSCs( G-4:6.10±0.23 vs G-1: 4.88±0.37; G-2:5.13±0.34; G-3:4.78±0.27,p<0.05) . Collagen content was reduced (G-l:40.86±4.82vsG-2:32.93±3.15;G-3:33.07±6.95;G-4:27.57±4.14,p<0.05) , and AP-MSCs had the predominant effect. (G-4 vs G-2; G-3,p<0.05) conclusion1, MSC transplantation improved cardiac function after MI.Cardiac functional benefits from intramyocardial transplantationof stem cells were associated with increased regional blood perfusion(angiogenesis),reduced the amount of the fibrosis and apoptotic cardiomyocytes,prevented post-infarction ventricular remodeling.2, AP- MSCs was superior to MSCs in cardioprotection at 4 weeks after transplantation3, MSCs mediate their protection partly by paracrine mechanism under ischemic conditions.
|
PDF Full Text Request