| Background: Coronary artery disease (CAD) is a common illness among middle-aged and elder people, and seriously influences the quality of life and health of these patients, even leads to death. In our country, with the improvement of people's standard of living and the change of food ingredient, the incidence rate of Coronary artery disease (CAD) or myocardial infarction (MI) is going up. A large area myocardial infracting may lead to cardiomyocytes loss and consequently deteriorate of heart function. Cardiomyocytes have a severely limited capacity for dividing new cardiomyocytes , though only a very small amount of myocardial cells could divide after infarct, it is not enough to recover damaged myocardium. At last, the fibroblasts repair it and shape scar tissue. Left ventricular remodeling will aggravate the patient's heart function and lead to heart failure, even to death. Clinical general treatments are drug treatment, interventional therapy, laser drilling in myocardium, CABG and heart transplantation, and so on. These methods could increase the blood supply of myocardium, but not to retrieve the necrosis myocardium, and have some contraindications and complications respectively. The therapeutic efficacy of CAD is not satisfied in clinical. In recent years, the reconstruction damaged myocardium is coming true, with the progress of the research on cell transplantation therapy, gene therapy, bioengineering materials. Many experiments reveal that various cells, including skeletal myoblasts,fetal cardiomyocytes, embryonic stem cells, bone marrow mesenchymal stem cells, and so on, are transplanted into damaged hearts and show benefits on heart functions. The disadvantage of cell transplantation is the small numbers of cells, the low viability of transplanted cells and the low residence rates, therefore, the therapeutic efficacy was not satisfied .With the development of cytology and tissue engineering technology, constructing engineering heart tissue in vitro is being realized. Looking for a new way to construct ideal engineering heart tissue is becoming a hot research point. MSCs are nonhematopoitic multipotent stem cells that exist in bone marrow over the whole lifespan of mammals. MSCs could differentiate to myogenic cells and be obtained by a simple routine of bone marrow aspiration. Autologous bone marrow cell transplantations have not immuno-rejective reaction. So they are ideal donor cells for seeding to infarction myocardium area. Degradable biomaterials, such as polyglycolic acid (PGA), polylactic acid( PLA) and their copolymer polylactic -glycolic acid (PLGA), are the implantable polymer materials studied and applied widely now. These materials could be degraded into CO2 and H2O, not accumulate in vivo, hardly any toxic side effect.The studies were designed to investigate the possibility inducing MSCs of rat differentiate into cardiomyocytes-like in vitro and the tissular compatibility by transplanting after MSCs adhering on PLGA in vivo.Objective: 1.To explore a method for the isolation, culture, purification and identification of MSCs and observe the biological feature of MSCs in vitro; Induce MSCs differentiate into cardiomyocytes-like with 5-aza in vitro and observe the change of MSCs appearance and identify the inducing results. 2.Take MSCs seed into PLGA and observe MSCs growing on PLGA; Implant PLGA with MSCs growing into homologous rat and observe the tissular compatibility between PLGA and the recipient. Our purpose is to provide the foundation for making the engineering heart tissue (EHT) in vitro. Methods:PartⅠ: the isolation, culture, purification and identification of rat MSCs and the fixing differentiation into cardiomyocytes-like in vitro. MSCs were obtained by pushing the morrow of adult SD rat's tibia and femur out with a syringe and isolated by gradient centrifugation method. Then the MSCs were purified and expanded though passage in vitro culture. Its surface antigen CD44 was detected with the method of immunohistochemistry. The third passage MSCs were respectively treated with 5μmol/l and 10μmol/l of 5-aza for 24h and observe the change of MSCs appearance using inverted microscope, and identify the inducing results by detecting the differentiated cells antigen (Actin and Desmin) with the method of immunohistochemistry and the expression of cardiomyocytes-specific gene (GATA-4 and Nkx2.5) by the method of reverse transcriptase-polymerase chain reaction (RT-PCR). PartⅡ:Transplantation after Adhering on PLGA in vivo. Taking MSCs seed into PLGA and observe MSCs growing on PLGA , culture with a routine method and make the MSCs adhere PLGA dividing and proliferating. The MSCs growing on PLGA were observed by electron microscope or by light microscope after HE staining .Implant PLGA with MSCs growing into homologous rat and observe the tissular compatibility between PLGA and the recipient.Results: 1. The MSCS adhered to culture dish sparsely and the majority of the cells displayed a spindle-like shape and took the appearance of fibroblasts. About 10d late, the bottom of culture dishes may be covered by MSCs completely. After digestion and passage 2h, MSCs began to adherence, and be spherical or ellipse. Then spread to the spindle-like or triangle-like shape, and gradually change into short fusiform shape or long fusiform shape, and bespread the dish bottom in 6-7d. The appearance and proliferation of MSCs didn't change, after cultured continuously at least 10 generations. Then the MSCs were purified and expanded though passage in vitro culture. Its surface antigen CD44 was detected with the method of immunohistochemistry. The third passage MSCs were induced with 5μmol/l and 10μmol/l of 5-aza in vivo, and 2w late the appearance of MSCs began to change, then the third passage by 5-aza for 4w gradually increased in size and formed a ball-like or stick-like appearance, and connected each other with adjoining cells, and began to form myotube-like structures. The differentiated cells antigen (Actin and Desmin) were positive with the method of immunohistochemistry and the expression of cardiomyocytes-specific gene (GATA-4 and Nkx2.5) were positive by the method of reverse transcriptase-polymerase chain reaction(RT-PCR).There were no beating cardiomyocytes-like in the whole induction process, but small amount lipocytes-like, neurocytes-like and osteoblasts-like could be seen. The results induced by 5μmol/l and 10μmol/l of 5-aza in vivo were similar. 2. The MSCs may adhere to PLGA dividing and proliferating. Implanting PLGA with MSCs growing into homologous rat, there were no rejection reaction between PLGA and the recipient, and the tissular compatibility was good.Conclusions: 1.The MSCs could be effectively isolated, purified, and expanded by density gradient centrifugation and adhering the culture dish; The MSCS cultured with 5-aza can differentiate into cardiomyocytes-like in vitro,but a few cells beyond the appearance of cardiomyocytes-like can be seen ,too. The differentiated mechanism is incompletely understood. 2.The MSCs may adhere to PLGA dividing and proliferating. There was a good tissular compatibility between PLGA and the recipient and. The cells implanted could grow well in vivo.
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