| Objective: 1.To implant the labeled BMSC in vitro in cardiac tissue of the DCM mice , it can be observed how BMSC differentiates and proliferates in cardiac tissue and how it influences on receptor's cardiac function, and we detect the mechanisms of such influence primarily.2.To inject EPO in abdominal cavities of the DCM mice, its influence on cardiac function of the diseased mice can be observed after injection, and detect the mechanisms of such influence primarily.3. The results of the two above experiments can be compared and analyzed.4. To compare the results and feasibility of the two kinds of DCM mice models induced by ADR and ISO respectively, we can provide some references of model-making methods that coincide with experimental principles and are operated more easily.Materials and Methods : 1. We used ADR (abdominal injection, 2. 5mg/kg/time, three times a week, two weeks in all) and high dose of ISO (85mg/kg/time, once a day, three days in all) to make DCM mice models, then compared the mortality, the changes of cardiac function and the results of histopathological sections between the two kinds of models, and summarized the differences of inducible methods and sickening characters between ADR and ISO, at last we chose the ADR inducible program to do the next experiment. 2. The DCM mice induced by ADR successfully were divided into four groups randomly.①BMSC transplantation group (Group A, n=10),②EPO therapeutic group (Group B, n=10),③control group (Group C, n=10),④sham operation group (Group D, n=10). In Group A, we used 100ul of BMSC suspension (5×10~6 cells included) to inject in left ventricular anterior wall in six separate spot, while the same amount of normal saline was used in the same spot in Group B and Group C,just underwent surgery but not injection in group D. after operation, we used 2000IU of human recombined erythropoietin (rhEPO) (1ml) to inject in abdominal cavity of each mouse in Group B (once a day, three days in all) while the same amount of normal saline was used in Group A and C, just puncture but no injection in Group D. Four weeks later, left ventricular ejection fraction (EF), left ventricular FS, left ventricular end systolic diameter (LVDs), left ventricular end diastolic diameter (LVDd), left ventricular systolic peak pressure (LVSP), left ventricular end diastolic pressure (LVEDP) and left ventricular maximal ascending or descending rate (+dp/dtmax, -dp/dtmax) in each group were detected by echocardiogram and physiological multichannel recorder. We used general pathological methods and ultraelectronicmicroscope to do histopathological detection, apoptosis index was detected by TUNEL method, apoptosis associated protein such as Bcl-2, Bax and Fas were detected by Western blot, VIII factor associated antigens were detected by immunohistochemical staining. We identified implanted cells and checked their locations with immunofluorescence double staining. The results could be compared among the four groups. Results: 1.The DCM mice models induced by ADR and ISO respectively were coincident with changes of DCM, according to the results of echocardiogram and hemodynamic detection and pathological and electronicmicroscopic examinations. In the process of making models, the mortality in ISO inducible group was a little higher than that in ADR inducible group (34. 28% vs 22. 86%).2. In Group A, it was observed by immunofluorescence that the implanted BMSCs could be alive and differentiate into cardiocyte, and be in regional distribution in cardiac tissue.3. Compared with Group C and Group D, EF, FS, +dp/dtmax, -dp/dtmax and LVSP in Group A and B all increased significantly (P<0.05), LVDs, LVDd and LVEDP all decreased significantly (P <0.05). Between Group C and D, EF, FS, LVSP, +dp/dtmax, -dp/dtmax, LVDd, LVDs, LVEDP had no significance of difference (P>0.05).4. Compared with Group C and D, apoptosis index in Group A and B decreased significantly, the expressions of Bcl-2 (anti-apoptosis protein) increased significantly while the expressions of Bax (pro-apoptosis protein) decreased.5. Compared with Group C and D, the number of immunohistochemical staining VIII factor positive cells in Group A and C increased significantly.6. Compared with Group C and Group D, the pathological changes and the cellular ultrastructural injuries in Group A and C improved significantly, the density of capillary increased.Conclusions : 1. Both ADR and ISO can induce the DCM mice models successfully. We find that ADR inducible program can be operated more easily, the cumulative myocardium toxicity of ADR progresses slowly, which can be more coincident with experimental principles.2. Injected in myocardium directly, the implanted BMSC in cardiac tissue can be alive and differentiate into cardiocyte. The new born of myocardium is mainly characterized by multiple cell homing, aggregation, fusion and differentiation.3. In the DCM mice models induced by ADR, BMSCs transplantation and EPO interference can improve cardiac function effectively . 4. In the DCM mice models induced by ADR, BMSCs transplantation and EPO interference can inhibite cardiocyte apoptosis and improving hyperplasia of capillary in cardiac tissue in the damaged region.5.In order to improve cardiac function of DCM induced by ADR, BMSCs transplantation is more effective than EPO interference.
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