Experimental Studies Of HUCB-MSCs Transplantation Into Rats With Dilated Cardiomyopathy

Objective:Adriamycin was administered intraperitoneally in Wistar rats for achieving DCM. To culture amplify of mesenchymal stem cells from human umbilical cord blood, cells were transplanted into the treatment group via the tail vein after identified as MSCs with flow cytometry (FCM). In order To investigate the HUCB-MSCs Engraftment in myocardium and the effects of grafted human umbilical cord blood mesenchymal stem cells (HUCB-MSCs) on LVEF,Plasma BNP and myocardium Pathological scores in rats with dilated cardiomyopathy (DCM). The expression of cardiac Troponin T(cTnT) of HUCB-MSCs in cardiac tissue was measured by immunohistochemistry and Immunofluorescence. To explored the mechanism of tail vein grafted human HUCB-MSCs which have benefical effects on cardiac function in rats with DCM.Method:100Wistar rats were randomly assigned into normal control group(normal group, n=10) and experimental group (n=90). Rats of experimental group were established dilated cardiomyopathy models according to the literature. The surviving rats the experimental group (n=46) were randomly diveded into two groups, DCM HUCB-MSCs treatment group (treatment group, n=23), DCM control group (control group, n=23). Human umbilical cord blood was collected from healthy maternal under aseptic technique conditions. Mononuclear cells of Human umbilical cord blood were isolated by lymphocyte separation medium using the Density-Gradient Technique. Then primary cells were incubated in a homeothermia culture chamber, HUCB-MSCs number and the rate of living cells were calculated through trypan blue staining, cells at the passage3were identified with flow cytometry (FCM) and labeled with Brdu, then calculate the labeling index (positive rate). Brdu-labeled P3cells were transplanted into the treatment group via the tail vein in twice, interval time were2weeks. Four weeks after transplanation, hearts were analyzed by morphological changes via HE staining and Pathological scores was calculated, LVEF was evaluated with the use of Echocardiography, BNP was measured with ELISA. Cardiac Troponin T(cTnT) was investigated by immunohistochemistry and LSCM to identify the expression in HUCB-MSCs.Results:1. HUCB-MSCs' flow cytometry and Brdu labeling results:The living rate of HUCB-MSCs at the third passage which Cultured using the Density-Gradient Technique through trypan blue staining is>95%. HUCB-MSCs at the third passage displayed high levels of mesenchymal cell's relative surface antigen marks such as CD29, CD105et.al as detected by flow cytometry and immunophenotypes, the rate is93.37%and89.08%. Furthermore, hematopoietic cell marks such as CD34, CD45are weakly expressed in HUCB-MSCs. the rate is0.89%and1.06%.87%HUCB-MSCs were labeled after72h and when the concentration of Brdu is10μmol/L2. Changes in Myocardium Pathological scores, LVEF and Plasma BNP before and after treatment:Four weeks after transplantation:①myocardium Pathological scores showed:the Pathological scores in treatment group was(0.82±0.37), compared with (1.17±0.81) of pre-treatment with significant differences, P<0.05; compared with (1.62±0.64) of control group with significant differences, P<0.05; compared with (0.12±0.04)of the normal group with significant differences, P<0.05;②LVEF measured by Echocardiography:the LVEF in treatment group was (62.36±4.72), compared with (40.02±2.04) of pre-treatment with significant differences, P<0.05; compared with (32.52±4.42) of control group with significant differences, P<0.05; compared with (78.14±3.64) of the normal group with significant differences, P<0.05;③BNP:the Plasma BNP in treatment group was (364.22±30.27), compared with (513.35±27.33) of pre-treatment with significant differences, P<0.05; compared with (592.58±35.74) of control group with significant differences,P<0.05; compared with the (76.03±5.27) of normal group with significant differences, P<0.05. 3. HUCB-MSCs' colonization and differentiation in the myocardium detected by Immunohistochemistry and LSCM:①Brdu-positive cells by Immunohistochemical staining can be seen in the treatment group. Immunohistochemical double-labeled in treatment group were seen scattered Brdu positive cells, yellow-brown cytoplasm of fiber structure can be seen in those Brdu Positive cells.②the expression of cTnT of HUCB-MSCs in myocardial tissue of treatment group Detected under LSCM, Myocardial cells differentiated from Brdu labeled HUCB-MSCs can be seen scattered in the Myocardial tissue, Brdu labeled HUCB-MSCs (red), expression of cTnT(green), The nucleus is blue.Conclusion:1. Cells which isolated from Human umbilical cord blood by lymphocyte separation medium using the Density-Gradient Technique incubated in a homeothermia culture chamber can harvest sufficient quantities of reliable cells, Cultured Cells and marrow mesenchymal stem cells were in the same of phenotypic expression.2. Transplanted HUCB-MSCs can significantly improve the myocardial pathological damage and function, increase LVEF, decrease Plasma BNP.3. HUCB-MSCs can migrate into Myocardial tissue of rats with DCM through vein transplantation and able to survive and Express cardiac-specific protein cTnT, which suggesting that HUCB-MSCs may differentiate into cardian-like cells.4. the HUCB-MSCs differentiation into cardiomyocyte-like cells may be one of the mechanisms, which improved cardiac function of DCM.