基于连续切片的兔心传导系统数字三维重建及起搏通道立体定位

The arrhythmia will dicrease the life quality, even cause cardiac sudden death,that is one of the most common disease.The disorder of the structure and function of cardiac conduction system, that is the most common reasons to cause the arrhythmia. The relationship between the structure changes and disorder of cardiac electricity physiology cause more and more attention, following the development of arrhythmia treatment work.It is meaningful significant to clarify the three dimensional anatomy and the microstructure of cardiac conduction system.. We choose the New Zealand rabbit whole hearts in different embryo age as the samples, cutting it for the preparation of series semithin sections, and using NF-160 labelled. We detect the positive part within the semithin sections under light microscope. At the same time, we prepare the series frozon sections of the whole heart, using HCN4 labelled, and detect the positive cells under the Laser Confocal microscope. Then we will get two series digital imagings about NF-160 positive part and HCN4 positive cells.The next, we use the RandAlign soft and AutoQuant three dimensional imaging system to track and pile up those digital imagings, and can get the three dimensional microimagings bank of cardiac conduction system.Then compare the positive part of the two sets images, to clarify the development process and the rules of development of the cardiac conduction morphology, and the distribution of the pacemaker gallery and the arrange of the pacemaker gallery and the around tissue. To explore the pathology mechanism of arrhythmia, establish the theory basis, provide the new thought and theory basis for prevention and cure arrhythmia.

心传导系统结构、功能改变引起的心律失常是常见病，轻者降低患者生活质量，重者导致心源性猝死。随着心律失常治疗工作的广泛开展，心传导系统结构改变与心电生理特性的关系逐渐引起了人们的关注，阐明其三维解剖关系及微细结构，对探讨心律失常的发病机制及其防治意义重大。本课题选用不同胚龄新西兰兔整心进行连续半薄切片、心传导系统细胞特定抗原NF-160免疫组化染色后光镜检测阳性部位，及连续冰冻切片、起搏通道HCN4免疫荧光染色后激光共聚焦显微镜检测阳性表达细胞，分别获取数字化显微图像；再应用RandAlign图像配准软件及AutoQuant三维成像系统，对图像进行追踪、叠加，得到心传导系统发育过程三维立体的微细结构影像图库,阐明其微细结构形态演变规律；再比对两套图片阳性区域，探讨起搏通道在心传导系统中的立体定位情况及与周围组织排列关系。为探讨心律失常发病机制奠定实验基础，为防治心律失常提供新思路和理论依据。

本研究采用组织学、连续切片、免疫荧光染色及分子生物学方法，观察不同胚龄胎鼠及成年小鼠心脏起搏组织具体定位，起搏细胞形态、数量及分布，并研究起搏细胞特异性标志蛋白NF-160、HCN4的表达部位及水平；以及探讨了起搏样细胞的诱导分化方法。发现，孕13天胎鼠右心房左背侧心外膜中可见到起搏组织雏形，境界不甚清晰，形状不规则，未见到典型窦房结动脉，其内细胞大小、形态不一。普通心肌细胞高表达Cx43，不表达HCN4；而该区细胞则相反，低表达Cx43，高表达HCN4，符合起搏细胞特征；同时在相邻的心内膜中可见到一些Cx43高表达、HCN4低表达细胞与该区细胞相连，推测在E13时，部分心内膜的细胞也具有HCN4离子通道。成年小鼠窦房结位于界沟和上腔静脉之间的心外膜处，位置偏上，略高于右心房顶，周围被结缔组织包绕，断面形态不规则，窦房结动脉穿行于窦房结组织中，在窦房结最下端可见窦房结细胞与心内膜浦肯野纤维相移行。窦房结内主要有起搏细胞、移行细胞和间质细胞；起搏细胞多位于中央，体积较大、核大，核染色浅，边界不清，胞质染色浅淡。移行细胞多位于周边，体积较起搏细胞小，染色较起搏细胞深。起搏细胞和移行细胞均表达NF-160和HCN4，而间质细胞二者都不表达。比较不同时期心房组织起搏相关基因表达水平，发现胚胎13天胎鼠心房HCN4表达水平远高于成鼠，Cx43表达水平与成鼠相似，而HCN2表达水平则远低于成鼠。结合胎鼠和成年小鼠窦房结位置变化，不难发现，窦房结的位置由最初的右心房左背侧向右上后迁移，最后定位于紧邻上腔静脉的心外膜结缔组织中。变化时限大致是从胚胎第10-11天到生后第5-7天。已将获取的连续切片光镜、免疫组化及激光共聚焦数字化图片建立完整的图像库，并将用于窦房结重建。已经撰写并发表论文3篇，其中1篇为SCI收录论文。还有一篇英文论文处于外投阶段。中国解剖学会2015年学术年会发表论文文摘一篇。还有部分实验数据正在整理，预计可再发表1篇英文论文。