Preliminary Exploration Of Construction Of Cardiac Atrioventricular Electrical Conduction Pathway By Rabbit Autologous Bone Marrow Mesenchymal Cells

Background and objective:Bone marrow derived mesenchymal stem cells (BMSCs) is another group of stem cells in bone marrow besides hematopoietic stem cells (HSC). BMSCs can differentiate to a variety of mesenchymal tissue, including bone, cartilage, fat, and even muscle tissue. It plays great effects in gene engineering and tissue engineering. All kinds of cardiomyocytes hardly regenerated when they were damaged. The myocardial depended on fibrin tissue to fix, which often led the damnification of pacing and conduction system, or decline of heart function. Traditional therapy ways would not bring into play good effects. When the disease of heart went into end-stage, the best way was heart transplantation. However, sources of donor were serious lack, and the patient must pay more money to buy immunosuppressive drugs postoperative for a long time. In recent years, with the depth of stem cell research, stem cell transplantation for regenerative therapy for heart disease has become a research hotspot. The use of BMSCs characterized by its own combination of genetic engineering research has made gratifying dawn side in the biological pacemaker. Some researches have tired to use BMSCs to cure heart conduction block and have demonstrated it broad application prospects. In our research, the rabbit BMSCs could differentiate cardiac-like cells by 5-aza induction, and the cell could express Cx43 and form Gap junction intercellular communication (GJIC) with cardiomyocytes in the early time. The autologous BMSCs were transplanted to atrioventricular suture area to construct a new channel of electrical conduction. We would use the general cardiac electrophysiology ways to check the bypass. It was beneficial that the BMSCs constructed cardiac electrical conduction pathway to the further study of the correlation of BMSCs with cardiac electrophysiology and the application of BMSCs in the bradyarrythmia.Materials and methodsThe BMSCs were separated and cultured from New Zealand rabbit bone marrow by discontinuous Percoll separation medium density gradient centrifugation and adherent culture method. Cardiomyocytes were separated and cultured from Westar neonatal rat heart by improved differential adhesion method. The BMSCs were expanded from primary generation to the forth generation in vitro, and were induced by 5-aza. Inverted microscope was used to observe differentiation of the BMSCs and morphology of the cardiomyocytes. Immunofluorescence was used to observe the changing of Cx43 in the BMSCs before and after the induction. The cardiomyocytes was as a positive group to check the expression of Cx43 between the BMSCs and the cardionmyocytes. We used this way to observe the GJIC formation between the BMSCs and the cardionmyocytes indirectly. Flow cytometry (FCM) was used to detect the differences of Cx43 expression in the BMSCs before and after induction. Scratch test was used to detect the change of GJIC in the BMSCs before and after induction. The firstly, the rabbits were cut the left edge of the sternum and the hearts were exposed. Fresh wound were made on the back of atria and left ventricle wall by scalpel, and Left atria and left ventricle of the rabbits were sutured by thoracotomy. One month after the surgery, the rabbits of experimental group (n=8) were cut the breast the secondly. The BMSCs which induced by 5-aza after four weeks and marked by DAPI before surgery were transplanted into the suture area, the DEME were used to replace BMSCs in the control group (n=6). One month after the transplantation, general cardiac electrophysiology testing ways were used to detect the existence of the bypass which formed by the BMSCs, and tested the characteristic of the bypass. Related technologies such as histopathology and immunoflurescence were used to observe the characteristics and survival of the BMSCs in the artrioventricular suture area and normal myocardial.Conclusion(1)Un-induced BMSCs could express Cx43 spontaneously, and the expression enhanced after induction by 5-aza. Cx43 expressed between BMSCs and cardiomyocytes which indirectly indicated formation of GJIC between tow kinds of the cells.(2) GJIC of BMSCs enhanced as the enhancement of Cx43 expression in the BMSCs after induction, which was the basic of constructing the new channel of cardiac electrical conduction.(3)The BMSCs could differentiate cardiomyocytes in the transplantation area, however, the survival of the BMSCs in the suture area was lower than in the normal cardiac tissue. In our research, two rabbits in the experimental group were indicated the possibility of the bypass existence by general cardiac electrophysiology testing. But there were not similar results in the control group.