[Expression of connexin 40 and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 in rat bone marrow mesenchymal stem cells cocultured with sinoatrial node tissues in vitro]

Abstract

Objective: To investigate the expression of connexin 40 (Cx40) and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) in rat bone marrow mesenchymal stem cells (BMSCs) cocultured with the sinoatrial node (SAN) tissues in vitro, so as to evaluate the possibility of BMSCs differentiation into SAN cells.

Methods: BMSCs were isolated from Sprague Dawley rats (aged 4-6 weeks, male or female) by the adhesive method and cultured; BMSCs at the 3rd passage were marked with carboxyfluorescein succinimidyl ester, and then were incubated on 6-well culture plate; cell climing slices were prepared at the same time. SAN tissue was taken and cut into 0.3 cm x 0.3 cm mass, and then placed into 4 degrees C PBS solution. The SAN tissue mass was cocultured with marked BMSCs at the 3rd passage for 3 weeks as the experimental group, and BMSCs at 3rd passage were cultured alone for 1 week as the control group. At 1, 2, and 3 weeks after coculture, the mean integrated absorbance (MIA) values of Cx40 and HCN4 were measured by Image pro plus 5.0 through the method of immunohistochemistry, and the mRNA expressions of Cx40 and HCN4 were identified by real-time fluorescent quantitative PCR.

Results: The MIA values of Cx40 and HCN4 in the experimental group were higher than that in the control group, showing significant differences (P < 0.01). In the experimental group, the expressions of Cx40 and HCN4 increased gradually with time. The longer the culture time was, the higher the expressions of Cx40 and HCN4 were, showing significant differences (P < 0.05). The mRNA expressions of Cx40 and HCN4 in the experimental group were significantly higher than those in the control group (P < 0.01); in the experimental group, the mRNA expressions of Cx40 and HCN4 increased gradually with time, showing significant differences between different time points (P < 0.05).

Conclusion: The expressions of Cx40 and HCN4 increase obviously after coculturing BMSCs with SAN tissue, indicating that BMSCs could differentiate into SAN cells by coculturing with SAN tissue in vitro.