S1P通过Cx43对成肌细胞移植治疗心肌梗死后心律失常的作用和机制研究

This research is the continuity of previous project funded by National Natural Science Foundation, based on previous studies and our research, connexin 43 (Cx43) regulates myocardial electrical homogeneity to reduce cardiac arrhythmia. Our hypothesis is that sphingosine 1-phosphate (S1P) regulates Cx43 through Ca2+，SERCA2a and microRNA-1 (miR-1), decreased cardiac arrhythmia after myoblast transplantation on myocardial infarction (MI). For the prevention and control of the arrhythmia after myoblasts transplantation, using animal model of myocardial infarction in vivo, we transfer S1P into the myoblasts. Then cells were transplanted to animal model of MI. In vivo, using ECG, radio frequency, transmission electron microscopy, the patch clamp technique，calcium fluorescent probe, and nitrate reductase immunohistochemistry, RT-PCR, Western-blotting, immunoperoxidase staining, observe the incidence of arrhythmia, measure the function of the coupling between the transplanted cells and host cells, detect electrophysiological characteristics of cells, check space structure and phosphorylation level of Cx43, measure expression of miR-1 and NO of myocardial tissue. In vitro, we use coculture model of myoblasts and cardiomyocytes, by transmission electron microscopy, calcium fluorescent probe, and nitrate reductase immunohistochemistry, RT-PCR, Western-blot and the patch clamp technique detect the connections of two cells, check electrophysiological characteristics of cells,, measure expression of miR-1. Try to find the role and molecular mechanism of S1P on arrhythmia after myoblast transplantation treatment of MI. In order to provide new ideas and new methods of decreasing arrhythmia after myoblasts autotransplantation. It will provide a theoretical basis for myoblasts autotransplantation on treatment of MI.

结合大量文献和坚实的前期基础，延续前一个国家基金课题，以心肌电均一性为视角，沿着1-磷酸鞘氨醇(S1P)借助Ca2+通过NO、miR-1借导的通路调控缝隙连接蛋白Cx43(Cx43)作用于移植区细胞，减少成肌细胞移植治疗心肌梗死后心律失常发生的思路，朝着Cx43调节心肌电均一性减少心律失常的目标开展工作。以在体心肌梗死动物模型和离体成肌细胞和心肌细胞共培养模型为研究对象，采用心电图、射频记录、透射电镜、钙离子荧光探针负载、膜片钳技术（原位、全细胞检测）等针对性手段，并结合RT-PCR、Western blot等常规方法检测心律失常发生率、移植细胞与宿主细胞之间的功能耦合、细胞电生理特性、Ca2+浓度、SERCA2a表达、NO含量、miR-1表达、Cx43 mRNA和蛋白表达及磷酸化水平，探讨S1P减少移植后心律失常的作用及分子机制，为减少自体成肌细胞移植后心律失常发生提供新思路和新方法。

目的：探讨成肌细胞加入外源性1-磷酸神经鞘氨醇（sphingosine 1-phosphate, S1P）S1P共同移植治疗大鼠心肌梗死模型是为减少单独移植造成的心律失常。以在体心肌梗死动物模型和离体成肌细胞和心肌细胞共培养模型为研究对象，采用心电图、射频记录、钙离子荧光探针负载、膜片钳技术等针对性手段，并结合RT-PCR、Western blot等常规方法检测心律失常发生率、Ca2+浓度、SERCA2a 表达、NO 含量、Cx43 mRNA 和蛋白表达及磷酸化水平，探讨S1P减少移植后心律失常的作用及分子机制。结果：离体实验：1）新生大鼠心肌细胞在培养24h后开始贴壁生长，培养6d内细胞数量未见明显变化，培养2 d后倒置显微镜下见散在的具有搏动能力的心肌细胞，第3d天可见散在的心肌细胞相互间连接成合胞且可见细胞搏动, 4-5d天成同一节律的搏动。2）不同浓度S1P均减少共同培养心肌细胞和骨骼肌成肌细胞缺氧/复氧损伤后LDH和MDA含量，即可减轻复氧所诱导的细胞损伤。3）S1P抑制共同培养心肌细胞和骨骼肌成肌细胞缺氧/复氧损伤后细胞凋亡。4）离体成肌细胞和心肌细胞共培养缺氧/复氧损伤模型中，随S1P浓度升高，Cx43蛋白表达明显上升。在体实验：1）导入GFP-S1P的骨骼肌成肌细胞移植后，在上述指标方面均优于对照组（骨骼肌成肌细胞组、导入GFP的骨骼肌成肌细胞组）及未转染成肌细胞移植组：导入S1P的细胞移植组发生心律失常的时间不但显著延后，诱发比率明显降低，而且持续时间短于对照组(P < 0.05)。2）导入S1P的细胞移植Ca2+-ATPase活性高于单纯细胞移植（P<0.05）。3）移植24 h后同转入脂质体的骨骼肌成肌细胞出现了移植细胞凋亡的减少（P<0.01）心肌梗死面积明显下降。4）心功能明显改善（P<0.01）。结论：S1P可减少因复氧所诱导的共同培养细胞损伤，抑制细胞凋亡。S1P通过提升心肌缺血再灌注大鼠心肌组织Ca2+-ATPase活性和缝隙链接Cx43蛋白，从而减少细胞移植后室性心律失常的发生，为减少自体成肌细胞移植后心律失常发生提供新思路和新方法。