体外诱导小鼠骨髓CD45—细胞向窦房结起搏样细胞定向分化

温昱 李彬

[摘要] 目的 探讨体外诱导小鼠骨髓CD45-细胞向窦房结起搏样细胞定向分化的方法。 方法 免疫磁珠分选小鼠骨髓CD45-细胞，体外培养扩增后，采用添加了5-氮胞苷和小鼠窦房结组织培养上清液的诱导培养基培养，经免疫荧光染色检测起搏样细胞标志物神经丝蛋白-160（NF-160）和超级化激活环核苷酸门控阳离子通道（HCN）4表达，并采用qRT-PCR定量检测起搏基因NF-160、HCN4和HCN2表达。 结果 小鼠骨髓CD45-细胞经体外诱导培养后得到的分化细胞表达NF-160和HCN4；qRT-PCR分析显示，与体外培养的小鼠窦房结细胞比较，分化起搏样细胞NF-160和HCN4的表达升高，HCN2的表达降低。 结论 体外培养能够将小鼠骨髓CD45-细胞诱导分化为窦房结起搏样细胞，为构建生物起搏器种子细胞提供选择。

[关键词] 起搏样细胞；CD45；HCN4；NF-160；窦房结

[中图分类号] R329.2 [文献标识码] A [文章编号] 1673-7210（2015）06（b）-0009-04

Inducing directional differentiation of mouse bone marrow CD45-cells into sinoatrial node pacemaker-like cells in vitro

WEN Yu1 LI Bin2

1.Department of Histology & Embryology， Basic Medical College of China Medical University， Liaoning Province， Shenyang 110122， China； 2.Department of Sports Medicine， Affiliated Shengjing Hospital of China Medical University， Liaoning Province， Shenyang 110022， China

[Abstract] Objective To study method on inducing directional differentiation of mouse bone marrow CD45-cells into sinoatrial node pacemaker-like cells in vitro. Methods Mouse bone marrow CD45-cells were derived by using immunomagnetic beads system. After amplification in vitro， induced medium supplemented with 5-azacytidine and culture supernatant liquid of mouse sinoatrial node cells was used to cultivate. The expression on pacemaker-like cells markers of neurofilament protein （NF-160） and hyperpolarization activated cyclic nucleotide-gated potassium channel （HCN） 4 were detected by immunofluorescence staining. The expression of pacing genes （NF-160， HCN4 and HCN2） were detected by qRT-PCR. Results Differentiated cells of bone marrow CD45-cells after induced by culture in vitro expressed NF-160 and HCN4. qRT-PCR analysis showed， compared with cultured sinoatrial node cells in vitro， the expression of NF-160 and HCN4 were higher， while expression of HCN2 was lower. Conclusion The sinoatrial node pacemaker-like cells can be induced and differentiated from mouse bone marrow CD45-cells， and the cells will be one choice of ideal seeding cells for biological pacemaker building.

[Key words] Pacemaker-like cells； CD45； HCN4； NF-160； Sinoatrial node

心律失常是常见病，严重将危及患者生命。目前，对于严重心律失常、病态窦房结综合征患者，安装心脏起搏器是理想的治疗方法[1-3]。随着生物学及干细胞研究的发展，利用其相关技术逐步构建生物起搏器，对受损心传导系统的组织进行修复或替代，尤其是窦房结功能重建，可以使心脏的起搏和传导功能得以修复[4-5]。构建生物起搏器的种子细胞目前以干细胞为主[6-7]。由于骨髓源干细胞具有多向分化潜能，可自体获得，并能够在体外扩增，是比较理想的种子细胞，所以本研究采用5-氮胞苷联合窦房结细胞培养上清液体外诱导培养骨髓CD45-细胞，探讨其分化为起搏样细胞的可能性。……