| Dilated cardiomyopathy (DCM) is a clinical common cardiac disease. Its etiology may be related to infection, autoimmune, metabolic abnormalities and family history. It is mainly characterized by dilatation and systolic dysfunction of left or bilateral ventricular. It is histologically marked by myocardial cell hypertrophy, degeneration, mitochondrial function defect, and different levels of fibrosis. The prognosis is poor, and 5-10-year survival rate is low (30% to 40%). Up to now, there is no specific drug treatment and methods. The current therapy is to control heart failure, lessen symptom and prevent ventricular remodeling, including the use of medicines, such as angiotensin converting enzyme inhibitor (ACEI), diuretics, digoxin,βblockers, calcium antagonists and other drugs. As the poor prognosis of the DCM patients, heart transplantation is the most effective treatment of patients with DCM in advanced stage, but many problems have been encountered in clinical practice, such as donor source, immunological rejection and costs. So cardiologists have been looking for a new treatment method. Stem cell transplantation as a new treatment of DCM has became a hot research area in recent years. Bone marrow mesenchymal stem cells (BMSCs) are non-hematopoietic stem cells in the bone marrow with a variety of sources, drawing convenience, easy expansion in vitro, pluripotency, and possess a weak immune rejection, and can be autologous transplantation with no ethical problems, and can be genetically modified, and can differentiate into myocardial cells and endothelial cells and other advantages. So BMSCs have become ideal seed cells for cell transplantation to treat cardiovascular disease. The treatment of acute myocardial infarction using stem cell transplantation has unparalleled advantages compared with conventional treatment, while the researches about the therapy of non-ischemic cardiovascular diseases with the BMSCs are less. Meanwhile many problems still remains, such as the choice of seed cells, the quantity of transplanted cells, the path of transplantation, treatment time, the duration of BMSCs proliferation and survival in the recipient, and how to improve the differentiation capabilities of BMSCs, when to follow-up after treatment, security in clinical application, which are need to be solved.Objective1. To explore the effect of differentiation of BMSCs into myocardial cells in different inducing conditions in vitro.2. To establish a stable and reliable animal model with DCM for basic experimental research.3. To explore the effect of BMSCs on the therapy of DCM with different transplantation methods and its mechanismMethods1. BMSCs were isolated with the density gradient centrifugation combined with adherent method and cultured. The growth curve, cell cycle, phenotype of BMSCs were determined. BMSCs were induced to differentiate into osteoblasts and adipocytes. In vitro BMSCs were cultured in the medium supplemented with 5–azacytidine(5–aza) or serum of rat with DCM combined with 5–aza or cadiocyte lysate of rat with DCM. Then the morphological changes were observed and the expression of cardiac troponin was detected using the methods of immunocytochemistry and RT-PCR.2. Rats DCM models were established by the following method. Male Wistar rats were administered doxorubicin 2.5 mg / (kg ? week) by intraperitoneal injection for 6 weeks. Then rats were observed for 2 weeks after the withdrawal. While rats in the control group were given the same volume normal saline with the same exposure route. The body weight changes and mortality were recorded, and echocardiography was performed. In addition to, plasma brain natriuretic peptide (BNP) level was detected, left ventricular internal diameter was measured, and pathological observation, electron microscopy, Masson staining of myocardium were performed.3. BMSCs labeled with Brdu and differentiated cells derived from the BMSCs in the medium supplemented with the serum of rat with DCM combined 5–aza were transplanted using a single intravenous injection and repeated intravenous injection method. The rats were randomly divided into normal control group (12 rats), model control group (17 rats), treatment group 1 (12 rats) which represents the single intravenous injection with BMSCs , treatment group 2 (12 rats) which represents the repeat intravenous injection with BMSCs, treatment group 3 (12 rats) which represents the single intravenous injection with differentiated cells cultured with the serum of rat with DCM combined 5–aza, treatment group 4 (12 rats) which represents the repeat intravenous injection with the same cells as in the treatment group 3. The state and mortality of the rats were observed, echocardiography, BNP levels, pathological observation and Masson staining were performed. The distribution and differentiation of transplanted cells in the heart were traced with laser scanning confocal microscope. The expression of VEGF was detected using immunohistochemistry and RT-PCR.Results1. Most of BMSCs isolated with the density gradient centrifugation combined adherent method and cultured adhered to the wall with the colony growth. Passaged cells quickly adhered, proliferated rapidly, and almost no colonies were formed. About 6 - 7d later, the cells crowded the bottom of the flask with the spindle-shaped uniform distribution. After many passages, cells have a more uniform and orderly distribution of fibroblast-like. After passaged for consecutive 9 times, cells had no significant changes in morphology, no signs of aging. The passage cycle is 6-7d, but after the 9 passage, the proliferation of cells was slow and the passage cycle was extended. The Passage1, 3, and 5 cells were high proliferation, the growth curve shape of which were similar. The majority of the cells were relatively quiescent. BMSCs could differentiate into osteoblasts and to fat cells under particular induced conditions. After the phenotypes of BMSCs were determined by flow cytometry, they were CD44-positive andCD34-negative. All the three methods could induce BMSCs to differentiate into cardiocytes-like cells with the expression of troponin, among which the growth state of cells in 5–aza group was poor; the cells in the other two groups grew better. The positive-expression rate of cardiac troponin of differentiated cells in the serum of rat with DCM combined 5–aza group was highest.2.The bodyweight was significantly different between normal group and model group on the 8th weeks. Rat mortality rate in the model group was 16.7%. The echocardiography findings, plasma BNP, left ventricular internal diameter were significantly different between normal group and model group. In the normal group, the results of HE showed that cardiac muscle fibers arranged regularly, no myocardial fiber breakage, uniform and rich cytoplasm, normal cell space and no edema. The results of electron microscopy displayed myocardial fibers arranged regularly, the uniform size of mitochondria, no edema, intact membrane and normal density of crest. The results of myocardial Masson staining showed collagen distributing sparse and complete collagen fibers nets of the neighbor cells and pale staining. In the model group, the results of HE staining showed myocardial fibers disorganized, myocardial fiber breakage, widened cell gap, edema and vacuole degeneration. The results of electron microscopy showed myocardial fiber fracture, swelling mitochondria, vacuole degeneration, and crest depletion. The results of myocardial Masson staining showed increased collagen in myocardium, breakage and derangement of collagen fibers surrounding the myocardial cells.The CVF compared with the normal group had significantly different.3. In model group without cell transplantation, the state of rats were poor, and body weight did not increase, seven rats died, and the mortality rate was 41.2%. While the state of rats in cell transplantation groups were improved, the body weight increased, the mortality of rats in all the cell transplantation groups was lower than that in the model group. The mortality rate of rats in repeat intravenous injection group was lower than that in single intravenous injection group. The cardiac function with echocardiography 2 weeks after cell transplantation had no significantly difference (p> 0.05) between treatment groups and model group. The results of cardiac function with echocardiography and plasma BNP 4 weeks after cell transplantation were: the results of treatment group 1, 2, 3, 4 were significantly different compared with that in the control group (p <0.05); the results in treatment group 2 and 4- were significantly different from that in the model group (p<0.05); there were significant difference between the treatment group 1 and treatment group 2 (p <0.05); there were significant difference between treatment group 3 and treatment group 4 (p <0.05); there was no significant difference between the treatment group 1, 3 and model group (p>0.05); there was no significant difference between treatment group 2 and treatment 4 (p> 0.05). The distribution and cTnT expression of transplant cells in myocardial tissue using the laser confocal microscope showed that transplanted cells labeled with Brdu could be found in the treatment group 1, 2, 3, 4, which distributed disperse, fused with myocardial cell, and expressed cTnT. However, the numbers of the transplanted cells labeled with Brdu in repeated intravenous injection treatment group 2, 4 were more than that in the single intravenous injection treatment group 1, 3, and the expression of cTnT in cells was significantly increased. There was no significant difference between treatment group 1 and treatment group 3 and between treatment group 2 and treatment group 4. The results of HE staining showed that myocardial cells still arranged disordered, myocardial fiber breakage was alleviated in treatment group 1 and treatment group 3 compared with model group. However, some changes still remained, such as widened cell gap, cell edema and vacuole degeneration in treatment group 1 and treatment group 3.The result of HE staining displayed that myocardial cells arrangement were no obvious disorder, no significant cardiac fiber broke, cell gap widened, edema and degeneration occurred, which significantly were alleviated compared with that in the model group in treatment group 2 and treatment group 4. Masson staining results showed there was still more myocardial collagen, and collagen fiber was still broken and disordered in treatment group 1 and treatment group 3. While there was less myocardial collagen in treatment group 2 and treatment group 4 than that in model group. In treatment group 2 and treatment group 4, there was no significant collagen fiber breakage and disorderly arrangement, and expression of VEGF was higher in treatment group 2 and treatment group 4 than that in other groups by immunohistochemistry and RT-PCR.Conclusion1. A number of highly active purified BMSCs were isolated by density gradient centrifugation combined with adherent method. 2. In vitro the three methods can induce BMSCs to differentiate into cardiocyte-like cells, which include 5–aza, the serum of rat with DCM model combined with 5–aza, the myocardium lysate of rat with DCM.3. The induced effects were better in the medium supplemented with the serum of rat with DCM combined with 5–aza, the myocardium lysate of rat with DCM than that with 5–aza. The cells growth state and differentiation into cardiocyte-like cells were the best in the medium supplemented with serum of rat with DCM combined with 5–aza (5μmol / L).4. The method with doxorubicin 2.5mg/(kg ? week) for 6 weeks by intraperitoneal injection to establish rat model with DCM was high success rate, shorter cycle of establishing models and reliable, which can be used as preparing animal models for basic research.5. The vein transplantation was an effective way, repeated intravenous treatment can increase the number of transplanted cells, extend the time of add donor cells, and its effect was better than that in single intravenous treatment.6. There were no significant difference between BMSCs transplantation treatment and transplantation treatment of cardiocyte-like cells differentiated from BMSCs.7.In vivo BMSCs can differentiate into myocardial cells, which expressed cardiac-specific cTnT.8. BMSCs transplantation for treatment of DCM in rats can reduce mortality and improve cardiac function, inhibit myocardial fibrosis, increase expression of VEGF and improve ventricular remodeling. |
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