Experimental Study On Differentiation Of Bone Marrow Mesenchymal Stem Cells Flowing Co-culture With Normal And Damaged Cardiomyocytes In Vitro

Mesenchymal stem cells(MSCs) are one kind of stem cells withthe abilities to differentiate in various directions. Some in vivo and in vitro experiments have proved that MSCs can be induced to differentiate to be osteoblasts, chondrocytes, fat cells, myocytes, cardiac myocytes. MSCs can be easily obtained and cultured in vitro, and they can be autotransplanted to repaire injured organs,so they will be widely used in clinic in the future,and have became one kind of important stem cell material for the reparation and reconstruction of tissues. In this experiment, we obtained and cultured the MSCs from rabbit bone marrow in vitro, studied the differentiation of 5-Aza-induced MSCs flowing co-culture with neonatal rat cardiomyocytes in different ways and tried to find the mechanism. We also studied the differentiation of MSCs in the microenvironment established by adriamycin-damagedcardiomyocytes to establish a new cell source of cell transplantation for cardiomyopathy diseases. The study includes the following:1.The efficient and practical method for the MSCs from rabbits isolation and culture in vitro was established after the repeatedpratices. The MSCs were gotten by gradient centrifugation and the espressions of surface antigens were monitored by flow cytometer. The result showed good cell uniform, negative expression of CD34 and positive expression of CD44. So the purity of the MSCs is high.2. The differentiation of MSCs flowing co-culture with neonatal rat cardiomyocytes in different ways: Rabit MSCs were isolated and cultured in vitro. After induced eled by 5-Aza and labeled by DAPI, MSCs were cocultured with neonatal rat cardiomycytes damaged by adriamycin in two different ways: 1) Co-culture system in which MSCs attached to cardiomyocytes directly(Contacted group); 2)double-chamber system in which MSCs and cardiomyocytes were separately cultured in upper and lower chamber through Millicell, respectively(Millicell group). On the 14th,21st and 28th days, MSCs were identified by means of transmission electron microscopy technique and immunochemistry techniques. And the transdifferntiation rates in different groups were detected by flow cytometer. MSCs only induced by 5-azacytidine were regarded as control group. The results showed that on the 14 th day, individual DAPI-labelled MSCs were positive expression of a-sarcomeric actin and desmin in the Contacted group; on the 21st, 28th day, positive cells were detected among the MSCs in the co-culture groups and Control group. Electron microscopy revealed typical striation, centrally positioned neuclei, rich glycogen granules and myofilament. Compared with other two groups, there were more double-positive MSCs in the Contacted group, and the ultrastructures in the MSCs were more mature.3. The differentiation of MSCs flowing co-culture withneonatal rat cardiomyocytes damaged by Adriamycin in different ways: After induced by 5-azacytidine and labeled by DAPI, MSCs from rabbit bone marrow were cocultured with adriamycin adriamycin neonatal rat cardiomycytes in two different ways. MSCs were identified on the 14th,21st and 28th days. The results showed that on the 21st, 28th day, some of the MSCs in the Millicell group and Control group were positive for the expression of a - sarcomeric actin and desmin; and positive cells were detected among DAPI-labeled cells in the Contacted group. Electron microscopy revealed some MSCs possessed the some characters of myocytes. Compared with other two groups, there were more double-positive MSCs in the Contacted group, and the ultrastructures in the MSCs were more mature.The above results showe: The isolated and cultured MSCs from rabbit bone marrow have the ability of plasticit. They can undergo myogetic differentiation in miscroenviroment established by normal and pathological cardiomyocytes in vitro. And direct contact to cardiomyocytes is crucial for the tr...