Song Bo, Liao Bin, Yu Fengxu, Xia Zenglian
Department of Cardiothoracic Surgery, the Affiliated Hospital of Luzhou Medical College, Luzhou Sichuan, 646000, PR China.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2012 Feb;26(2):146-51.
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.
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.
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).
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.
研究体外将大鼠骨髓间充质干细胞（BMSCs）与窦房结（SAN）组织共培养后连接蛋白40（Cx40）和超极化激活环核苷酸门控阳离子通道4（HCN4）的表达情况，以评估BMSCs分化为窦房结细胞的可能性。
采用贴壁法从4至6周龄的Sprague Dawley大鼠（雄性或雌性）分离并培养BMSCs；对第3代BMSCs用羧基荧光素琥珀酰亚胺酯进行标记，然后接种于6孔培养板；同时制备细胞爬片。取SAN组织并切成0.3 cm×0.3 cm大小的组织块，然后置于4℃ PBS溶液中。将SAN组织块与标记的第3代BMSCs共培养3周作为实验组，将第3代BMSCs单独培养1周作为对照组。共培养1、2和3周后，通过免疫组织化学方法用Image pro plus 5.0测定Cx40和HCN4的平均积分吸光度（MIA）值，并用实时荧光定量PCR鉴定Cx40和HCN4的mRNA表达。
实验组Cx40和HCN4的MIA值高于对照组，差异有统计学意义（P<0.01）。在实验组中，Cx40和HCN4的表达随时间逐渐增加。培养时间越长，Cx40和HCN4的表达越高，差异有统计学意义（P<0.05）。实验组中Cx40和HCN4的mRNA表达明显高于对照组（P<0.01）；在实验组中，Cx40和HCN4的mRNA表达随时间逐渐增加，不同时间点之间差异有统计学意义（P<0.05）。
BMSCs与SAN组织共培养后Cx40和HCN4的表达明显增加，表明BMSCs体外与SAN组织共培养可分化为窦房结细胞。
