大鼠BMSCs体外诱导培养后连接蛋白40及超极化激活环核苷酸门控阳离子通道4表达的初步研究

目的观察大鼠BMSCs与窦房结组织块混合诱导培养后连接蛋白40(connexin 40,Cx40)及超极化激活环核苷酸门控阳离子通道4(hyperpolarization-activated cyclic nucleotide-gated cation channel 4,HCN4)的表达情况,探讨大鼠BMSCs向窦房结细胞诱导分化的可能性。方法 4～6周龄SD大鼠20只,雌雄不限,体重200～300 g。取6只SD大鼠,采用贴壁筛选法分离BMSCs,取第3代细胞以羧基荧光素二乙酸盐琥珀酰亚胺酯标记后,接种于6孔培养板内,同时制作细胞爬片。取14只SD大鼠窦房结组织,剪成0.3 cm×0.3 cm大小组织块,与标记后的第3代BMSCs混合培养,作为实验组;对照组仅单独培养第3代BMSCs。对照组培养1周及实验组混合培养1、2、3周,采用免疫组织化学方法检测Cx40和HCN4表达,并采用Image pro plus 5.0图像分析软件检测各组细胞表达Cx40和HCN4的平均积分吸光度值(mean integrated absorbance,MIA);采用实时荧光定量PCR检测细胞Cx40及HCN4 mRNA表达水平。结果免疫组织化学染色检测示,实验组混合培养1、2、3周,Cx40和HCN4 MIA值均显著高于对照组(P<0.01);且实验组随培养时间延长,Cx40和HCN4MIA值均逐渐增加,各时间点间差异均有统计学意义(P<0.05)。实时荧光定量.PCR检测示,实验组混合培养后1、2、3周,Cx40和HCN4 mRNA表达水平均显著高于对照组(P<0.01);实验组随培养时间延长,Cx40和HCN4 mRNA表达水平均逐渐增加,各时间点间差异均有统计学意义(P<0.05)。结论将大鼠BMSCs与窦房结组织块体外混合培养,诱导后细胞高表达Cx40及HCN4,具有向窦房结细胞分化的可能。

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

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×0.3 cm mass,and then placed into 4℃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.