| Abstract:objective:To investigate the expression of Cx40 and HCN4 in rat bone marrow mesenchymal stem cells (MSCs) co-cultured with the sinoatrial node (SAN) tissues in vitro, so as to evaluate the possibility that marrow mesenchymal stem cells differentiated into sinoatrial node cells.Methods:(1) Rat MSCs collection:The femurs and tibias of healthy SD rats were dissected after being drowned in 75% alcohol. Femurs and tibias bone marrow cavities were flushed by PBS solution. Then the cell suspension was collected into centrifuge tubes. (2) MSCs separation and culture:Centrifuged the cell suspension, discard supernatants, mixed the cells with DMEM containing 10% FBS and then inoculated the cells into the flasks. MSCs were separated and purified by differential velocity adherent technique, then cultured in incubator (temperature maintained 37℃and concentration of CO2 was 5%). When MSCs grew to cover 80% of the flask, they were digested by Trypsin and subcultured. (3) Identification the purity and activity of MSCs:The purity of the third generation of MSCs was identified by CD34, CD44 antibodies immunofluorescence method, and the cells activity was identified by Trypan blue dye. (4) MSCs inoculation:The third generation cells were marked by CFSE (5,6-carboxyfluoresceindia cetate, N-succinimidyl ester) to ensure that the cells used were derived from MSCs, and were inoculated at the same concentration (1×105/ml) into cultivating holes or on glass coverslips. (5) Rat sinoatrial node tissue collection:The SD rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The hearts were dissected under sterile conditions, washed repeatedly with PBS solution. Sinoatrial node tissue was taken and cut into 0.3cm×0.3cm mass, which were then placed into 4℃PBS solution. (6) Co-culture MSCs and SAN:The sionatrial node tissue mass were placed into cultivating holes to co-culture with MSCs but not touched to MSCs. (7) Classification:Control group (Group A):The MSCs were cultured for 1 week independently. Experiment group:The MSCs were cultured with the sinoatrial node tissue for 1 week (Group B),2 weeks (Group C) and 3 weeks (Group D). (8) Cell screening:The cells of higher labeled rate (>90%) by CFSE in each group were selected to ensure that all of the cells used for identifying the expresssion of Cx40 and HCN4 were derived from the 3rd MSCs. (9) Expression of Cx40 and HCN4:The cells on the coverslips were fixed with 75% alcohol, then the MIOD (mean integrated optical density) of Cx40 and HCN4 in cells in each group was measured by Image-pro plus 5.0 through the method of immunohistochemistry. (10) mRNA expression of Cx40 and HCN4:The mRNA expression of Cx40 and HCN4 in each group were identified by Real-time fluorescent quantitative PCR. (11) Analysis:All of the experimental data were analysed by statistical software SPSS 13.0. The ANVOA method was used for comparison between groups. Results:(1) The purity of MSCs identified by immunofluorescence method was more than 80%. The cells activity identified by Trypan blue dye was over 90%. (2) The MIOD values of Cx40 and HCN4 in experiment group were higher than the control group, the differences were significant (p<0.01). In experiment group, the expression of Cx40 or HCN4 increased gradually with time. The longer period the cells were cultured, the higher of the expression of Cx40 and HCN4 were (p＜0.05). (3) The mRNA expression of Cx40 and HCN4 in experiment group were higher than the control group (p＜0.01), and the difference were significant within the experiment group (p＜0.05). Conclusion:(1) In this study, the methods that we collected and separated the rat bone marrow mesenchymal stem cells by differential velocity adherent technique were reliable. When the cells spread to the third generation, we can attain more pure MSCs with more activity. (2) The expression of Cx40 and HCN4 increased obviously after co-culturing MSCs with sinoatrial node tissue. (3) Through this experiment, we found it was possible that rats MSCs could differentiate into sinoatrial node cells by co-culturing them with sinoatrial node tissue in vitro. |
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