The Experimental Study Of Treating Acute Myocardial Infarction By Adipose Tissue Stromal Cells Transplantation

PartⅠComparison of mesenchymal stem cells from adipose tissue and bone marrowbonearrowObjective To compare adipose stromal cells (ASCs) and bone marrow mesenchymal stem cells (MSCs) and make a foundation for cell therapy basing on ASCs. Methods Human adipose tissue was digested with collagenase typeⅠsolution and ASCs were derived by culture. At the same time, Mononuclear cells were isolated from human bone marrow and MSCs were derived by culture. Cells number was compared before and after culture. Two population of cells surface phenotype (CD13, CD29, CD31, CD34, CD44, CD45, CD49d, CD105, CD106 and HLA-DR) was examined by flow cytometry. These two kinds of cells were added to mixed lymocyte cultrures and PHA-induced lymphocyte transformation cultures with various concentratons. The proliferation of lymphocyte was messured by MTT method, effect of ASCs and MSCs on mixed lymphocyte response and PHA-induced lymphocyte transformation were investigated. At the same time, ASCs were cultured under the induction myocardial cell lysate, the expression of myocardium specific protein and genes after induction were tested by immunofluorescence method or RT-PCR.Result ASCs were isolated and cultured from human adipose tissue. The cultured ASCs displayed a fibroblast-like morphology and were similar to MSCs. Even the number of nucleated cells isolated from bone marrow was significantly higher than from adipose tissue(P<0.001), No significant differences were observed for yield of adherent stromal cells (P>0.05). Both cells expressed CD13, CD29, CD44, CD105 and both cells were negative for CD31, CD34, CD45 and HLA-DR. Difference in expression were noted for CD49d and CD106. The same number of ASCs and MSCs showed comparatively negative immunomodulatory functions by inhibited the mixed lymphocyte response and induction of transformation (P>0.05). After 2 weeks of culture under the induction of myocardial cell lysate, cells expressed cTnT and desmin as well as the myocardium specific genes cTnT, ANP andαMHC.Conclusion Like MSCs, ASCs displayed the similar fibroblast-like morphology. Yield of adherent stromal cells per gram of tissue was similar between bone marrow and adipose tissue. ASCs were similar to MSCs in the features of CD markers and immunosuppressive properties. ASCs could be differentiated into myocardial like cells induced by myocardial cell lysate. So human adipose tissue is an abundant and accessible source of adult stem cells. and this may make a foundation for the study of cell therapy basing on ASCs.PartⅡEffect of cytokines secreted by human adipose stromal cells on endothelial cellsObjective To isolate and culture adipose stromal cells (ASCs), and study the angiogenic or antiapoptotic effect of cytokines secreted by ASCs on endothelial cells.Methods Human adipose tissue was digested with collagenase typeⅠsolution and ASCs was derived by culture. The cultured cells surface phenotype (CD31, CD44, CD45 and CD105) was examined by flow cytometry. ELISA was used to detect the secretion level of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and stromal cell derived factor-1α(SDF-1α) and RT-PCR was used to detect the expression of their mRNA. Ehdothelial cells were isolated from human umbilical vein and cultured. Then the ASCs medium was used to culture human umbilical vein endothelial cells. Endothelial cells counting by hemacytometer to determined the proliferation. Endothelial cells were also incubated with 10ng/ml TNF-αin the presence or absence of ASC-conditioned medium. To assess cell viability, endothelial cells were harvested 24 hours later, stained with annexin V-FITC /propidium iodide (PI) and then subjected to analytic flow cytometry to assess the apoptois of endothelial cells. RT-PCR was used to detect the expression of Bcl-2 mRNA in the endothelial cells.Result ASCs was isolated from adipose tissue and derived by culture. Examined by flow cytometry, ASCs expressed CD44 and CD105 while did not expressed CD31 or CD45. Significiant amounts of cytokines were measured in the supernentant by ELISA. The levels of VEGF were (75±16) pg/ml, HGF were (637±157) pg/ml, SDF-1αwere (482±113) pg/ml. The mRNA of VEGF, HGF and SDF-1αwere significantly expressed in ASCs. Endothelial cells were cultured with or without ASCs medium. 3 days later the number of endothelial cells in ASC-conditioned medium (125±19)% was significantly higher than that of controls (97±12)%(P<0.05). After cocultured with TNF-α24 hours, the apoptosis rates of endothelial cells in ASC-conditioned medium (3.1±1.2)% was significantly lower than those of controls (6.9±1.7)%. (P<0.05). Conclusion ASCs can secrete cytokines and have significantly effect on the endothelial cells proliferation and apoptosis. PartⅢAdipose tissue stromal cells transplantation in rats of acute myocardial infarctionObjective The present study was to investigate the proliferation and differentiation of rat adipose stromal cells (ASCs) when implanted into ischemic myocardium and the improvement of heart function.Methods Sprague-Dawley rat adipose tissue was digested with collagenase typeⅠsolution and ASCs were derived by culture. The cells surface phenotype (CD31, CD44, CD45 and CD90) was examined by flow cytometry. ASCs labeled with 4'6-diamidino-2-phenylindole (DAPI, ASCs group) or Dulbecco's modified Eagle medium (DMEM, control group) was transplanted into the ischemic myocardium, which was produced by ligation of left descending branch of coronary artery. At 1 and 4 weeks after transplantation, specimens were acquired from infracted area and also, echocardiography was done to detect the effects on heart function. Then cell morphology and capillary density were measured, and vascular endothelial growth factor (VEGF) expression levels were assayed by RT- PCR and ELISA.Result ASCs derived by culture expressed CD44 and CD90 while did not expressed CD31 or CD45. ASCs were alive at 1 and 4 weeks after transplantation and had a trend toward differentiation into vascular endothelial cells. The number of capillary vessels in peri-infarct area in ASCs group increased significantly compared with control group (P <0.01). The levels of VEGF mRNA and protein expression at 1 week increased significantly in ASCs group compared with control group (P < 0.01). Left ventricular function, including ejection fraction, fractional shortening were higher in ASCs group when compared with control group at 4 weeks (P <0.01).Conclusion ASCs transplantation can accelerate angiogenesis in infarcted area after rat myocardial infarction and improve heart function.