| Ischemic heart disease is one of the leading causes of death and the incidence rate is increasing. Cardiac myocytes have been traditionally regarded as terminally differentiated cells and necrosis cardiac myocytes have no ability to regenarate. Recent studies have shown that the adult cardiac myocytes can split and proliferate, but the splitting index is only0.03%～0.9%[1-3]; several studies suggested that the resident cardiac stem/progenitor cells in myocardium can differentiate into mature cardiomyocytes, but the number of CSCs is too limited so they are not enough to generate myocardium and instead the infarcted heart. At last the fibrotic myocardium is instead of scars tissue, and the heart function of contraction is lost, aggravating to heart failure. Although heart transplantation is the effective approach for treating the patients with final heart disease, the donor is limited and the side effects of immunosuppressive agents can’t be ignore.The common treatments for ischemia heart diseases are pharmacotherapy, coronary artery intervention and coronary artery bypass grafting, which mainly focus on ameliorating myocardial ischemia, but little effect on necrosis myocardium. Thus many researchers contribute to exploring a novel approach for more benefits. Several reports showed that stem cells can differentiate into myocardial cells, smooth muscle cells or endothelial cells. and transplantation of stem cells can promoting angiogenesis in infarcted region and enhance heart function, representing a new strategy for cardiac repair. For the ethics problems and immune rejection, the researches and applications of embryonic stem cells are limited. The autologous BMSCs are better for its easy acquisition, high proliferation rate and null immune rejection. Researchers found that BMSCs possess the characterizations of complexity, pluripotency and migration.There is intense inflammation response in infarcted region after MI. Several cytokines are exist in infarcted region, such as SDF-1, MCP-1, The research of Ma et al.[6] suggested that the expression of SDF-1increased at MI early stage, and it promoted MSCs homing to MI region via SDF-1/CXCR4axis. Askari[7] reported that with the induction of SDF-1, the homing of G-CSF stimulated MSCs to MI region was enhanced markly.Myocardial infarction (MI) is one of the leading causes of death and the ischemic injury is usually irreversible despite aggressive medical and revascularization treatment.Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for diseases exclusively through hypertrophy.Recent studies have shown that the resident cardiac stem cells (CSCs) exist in heart and can differentiate into cardiomyocytes.smooth muscle cells or endothelial cells, and transplantation of MSCs can reduce infarcted size and enhance heart function, representing a new strategy for cardiac repair. It is still not fully understood how these MSCs are recruited into the injured myocardium after MI. Up to now,stromal cell-derived factor-la (SDF-1a) and its cellular receptor CXCR4(C-X-C chemokine receptor type4) are known as the most prominent stem cell chemotaxis.SDF-lahas been shown to be significantly upregulated in myocardial infarction and attract the CXCR4+stem cells towards SDF-1agradient. Thus, upregulation of SDF-1aexpression in ischemial myocardium represents a promising therapeutic strategy to improve post-infarction therapy.However,the role of SDF-1α/CXCR4pathway in MSCs migration needs to be further clarified. Phosphatidylinositol3-kinase (PI3K) is widely expressed and plays crucial roles in regulating multiple cell processes,such as cell migration, proliferation, differentiation, motility,survival and angiogenesis.Previous studies have demonstrated PI3K is also involved in SDF-1α/CXCR4-mediated chemotaxis and multiple stem/progenitor cells migration. The purpose of this study was to address the following questions:1) whether overexpression of SDF-1αresulted in MSCs migration and accumulation in the infarcted region, and2) whether SDF-la-induced MSCs migration was mediated via CXCR4/PI3K signaling pathway.1. Take the femur-tibia/fibula complex and using a scalpel separate the femur from tibia/fibula under sterile surroundings, remove the muscle tissues and wash the marrow cavity with PBS, centrifuge and suspense the cells pellet, culture BMSCs with adherence method.2.Insert the breathing tube to trachea and open the thoracic cavity between3,4ribs, ligate the LAD to construct the mouse MI model. Test the LVEF/FS/LVEDD to determine the MI model is constructed successfully.3. Culture/expand the BMSCs in vivo and sort the cells with magnetic micro beads to pure the CXCR4+/CCR2+cells. Test the BMSCs with FACS to identify the surface marker of sorting cells.4. Evaluate the effect on BMSCs migration of CXCR4/CCR2and explore the possible mechanism. Seed the myocardium cells in lower chamber and BMSCs in upper chamber, evaluate the migration of BMSCs to lower chamber. There are4groups in the test, control, CXCR4+/CCR2+, CXCR4+/CCR2-, CXCR4-/CCR2+. Count the cells in5high power filed at24h after incubation. Use immunofluorescence to detect the cells migration to lower chamber.5.3d After the MI mouse model constructed, inject the sorting cells to MI myocardium in5points, observe the effect of transplanted BMSCs on MI myocardium. There are6groups in this experiment:sham, MI, PBS, CXCR4+/CCR2+,CXCR4+/CCR2-, CXCR4-/CCR2+, CXCR4-/CCR2-.6. Detect the homing of BMSCs with immunofluorescence, choose10high power filed under fluorescence microscope randomly for the MI region of every heart sample, count the CM-DIL positive cells and make the statistical analysis.7.Apply western blotting assay to value the effect of PI3K/Akt pathway on the migration of sorting BMSCs. In the Transweli chamber model, MCP-1/SDF-1are added in the lower chamber. There are12goups in this test, control, CXCR4+/CCR2+, CXCR4+/CCR2-, CXCR4-/CCR2+, control(MCP-1/SDF-1), CXCR4+/CCR2+(MCP-1/SDF-1), CXCR4+/CCR2-(MCP-1/SDF-1), CXCR4-/CCR2+(MCP-1/SDF-1), control (MCP-1/SDF-1, LY2394002), CXCR4+/CCR2+(MCP-1/SDF-1, LY294002), CXCR4+/CCR2-(MCP-1/SDF-1,LY294002),CXCR4-/CCR2+(MCP-1/SDF-1、LY294002). Detect the expression level of pAkt via Western Bloting, MIO(the sample band)/MIO(internal control band) represents the intensity of pAkt expression. Make statistical analysis to value the effect of PI3K/Akt pathway on migration of CXCR4+/CCR2+cells. |
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