Combination Of Bone Marrow Mesenchymal Stem Cells Transplantation And Rosuvastatin For Treatment Of Coronary Microembolization In Rats

Coronary microembolization (CME) is a frequent and important event in acute coronary syndromes and during coronary interventions,which leads to bad prognosis and higher mortality for patients. But effective therapies have been limited so far. Here, we aim to copy a model of CME with homologous microthrombi and observe the curative effect of bone marrow mesenchymal stem cells ( BMSCs) transplantation in combination with rosuvastain for treatment of CME in rats.PartⅠIsolation,cultivation and identification of rat BMSCs in vitroObjective To establish a effective method of isolation, cultivation and identification of rat BMSCs in vitro. Methods BMSCs were seperad from the bone marrow of rats based on their adherence to tissue culture surface. The growth properties were compared by cell counting between successively passaged BMSCs( P1, P3, P5). P3 BMSCs were further identified by fluorescence-activated cell sorter (FACS) for CD11b,CD34,CD44,CD45,CD90 antibodies.Simultaneously, osteoblastic and adipocytic differentiation were also induced and observed. Results A gradual loss of replication ability was companied with BMSCs passaged from P1 to P5. P3 BMSCs were showed active proliferative ability and good purification.They were also showed CD44,CD90-positive and CD11b, CD34,CD45-negtive.Further, Osteoblastic and adipocytic differentiation were got positive. Conclusion The attachment culture method can be used to isolate and purify BMSCs of rats, which helps for further study.PartⅡThe effect of transplantation with bone marrow mesenchymal stem cells following coronary micoembolization in ratsObjective To copy the model of CME with homologous microthrombi and observe the curative effect of BMSCs transplantion for treatment of CME in rat. Methods pGC FU-GFP-LV, a lentiviral vector with green fluorescent protein(GFP)used as a tool, P1 BMSCs were infected at MOIs of 2, 8 and 32 respectively. The GFP-positive cells were detected by fluorescence microscope. After infected by the virus according to appropriate MOI, P1 BMSCs were passaged to P3 for transplantation. A total of 64 male Sprague Dawley(SD) rats were randomized into control group, CME group, low-dose BMSCs group(2×105) and high-dose BMSCs group (2×106) averagely. The model of CME was copied by injection of homologous microthrombotic particle suspension into left ventricle when clamping the ascending aorta. The rats were sacrificed at day 3,7,and 28 after operation. Surviving cells were tracked and their differentiation into cardiomyocyte-like cells was evaluated by immunofluorescent staining after transplantation. Expression of TNF-a, IL-1β, IL-4, IL-10 in myocardium were assay by Enzyme linked immunosorbent assay(ELISA), immunostaining and VEGF, bFGF were detected by RT-PCR, western blot. Pathological changes were detected by HE staining and angiogenesis was quantified by immunnostaining forⅧfactor antibody. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results More than 90% P1 BMSCs were GFP-positive at MOIs 8 and fluorescent intensity was no changes when P1 BMSCs passaged to P3. Compared with control group, higher expression of TNF-a, IL-1βin myocardium, multi-focal myocardial necrosis with inflammatory cells infiltration, fibrosis in subendocardial region and significant cardiac dysfunction were observed in CME group, indicating it was a successful model. No BMSCs survival were observed nearly at 28 days after transplantation in both of BMSCs groups . Whether high-dose BMSCs group or low-dose BMSCs group, the expression of TNF-a, IL-1β, IL-4, IL-10, VEGF, bFGF in myocardium were no difference when compared with CME group(p>0.05). Inflammatory cell infiltration, collagen deposition and capillary density in the farct zone in both of BMSCs groups were as same as in CME group. Further, recovery of cardiac function was also not found in both of transplantation groups when compared with CME group(p>0.05). Conclusion Lots of BMSCs may be lost in a few days after transplantation due to serious inflammation in myocardium following CME. BMSCs transplantation alone can not prevent cardiac remodeling and dysfunction in rats with CME.PartⅢThe effect of rosuvastatin for treatment of myocardial inflammation following coronary microembolization in ratsObjective To evaluate the effect of rosuvastatin on myocardial inflammation and cardiac dysfunction following CME in rats. Methods A total of 48 male SD rats were randomized into control group, CME group, low-dose rosuvastatin group(0.5mg/kg.d) and high-dose rosuvastatin group(3.0mg/kg.d) averagely. Rosuvastatin was dissolved into saline fed in perioperation for two weeks and the rats were sacrificed at day 3 and 28 after operation. Expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were assay by ELISA, immunostaining and pathological changes were detected by HE staining. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results The expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were no difference between low-dose rosuvastatin group and CME group(p>0.05).In contrast, the levels of TNF-a and IL-1βwere lower, and IL-10 was higher in high-dose rosuvastatin group compared with CME group(p<0.05). High-dose but not low-dose rosuvastatin also reduced inflammatory cell infiltration and collagen deposition in farct zone,decreased LVEDD,LVESD and increased LVEF,LVFS. Conclusion In rats with CME, perioperative therapy with high-dose rosuvastatin prevents cardiac remodeling and dysfunction. This benefit may be partly derived from reducing myocardial inflammation. PartⅣCombination of bone marrow mesenchymal stem cells transplantation and rosuvastatin for treatment of coronary microembolization in ratsObjectct To observe the curative effect of BMSCs transplantation in combination with rosuvastatin for treatment of CME in rats, and to compare with the effect of BMSCs transplantation or rosuvastatin alone. Methods A total of 48 male SD rats were randomized into control group, CME group, BMSCs group(2×10~6), rosuvastatin group(3.0mg/kg.d) and BMSCs+rosuvastatin group averagely. The rats were sacrificed at day 3 ,7 and 28 after operation. Surviving cells were tracked and their differentiation into cardiomyocyte-like cells was evaluated by immunofluorescent staining after transplantion. Expression of TNF-a,IL-1β,IL-4,IL-10 in myocardium were assay by ELISA, immunostaining and VEGF,bFGF were detected by RT-PCR ,western blot. Pathological changes were detected by HE staining and angiogenesis was quantified by immunnostaining forⅧfactor antibody. Collagen deposition was detected by Masson staining and recovery of cardiac function was assessed by echocardiography. Results BMSCs+rosuvastatin group increased about 45-fold of the cellular survival at 28 days when compared with BMSCs group(p<0.05). Only few transplanted BMSCs fusion with host cardiomyocytes but not differrention were detected. Expression of TNF-a,IL-1βin myocardium were lower,and IL-10 was higher in BMSCs+rosuvastatin group than in rosuvastatin group or BMSCs group(p<0.05). Expression of VEGF,bFGF were also higher in BMSCs+rosuvastatin group than in BMSCs group(p<0.05). Further, BMSCs transplantation in combination with rosuvastatain significantly inhibited inflammatory cell infiltration , collagen deposition in the infarct zone, decreased LVEDD,LVESD and increased LVEF,LVFS when compared with rosuvastatin group or BMSCs group alone. Conclusion BMSCs transplantation in combination with rosuvastatain significantly inhibites inflammation in myocardium when compares with rosuvastatain or BMSCs alone, enhances survival of BMSCs under ischemic condition., and significantly prevents cardiac remodeling and dysfunction after CME.