EMT信号在Tbx18+CPCs定向分化心脏起搏细胞中的调控作用

Uncovering the origin of pacemaker cells and the mechanisms of its differentiation is of great significance to the research of biopacemaker. Tbx18+cardiac progenitor cells (CPCs) is a progenitor cell populations derived from a sources of proepicardium, the pacemaker cells of sinoatrial node derived from this progenitor cells. The epithelial-mesenchymal transition(EMT) is closely related to the differentiation of proepicardial progenitor cells. However, whether the differentiation of pacemaker cells of Tbx18+CPCs is regulated by the EMT signal remains unclear. So, we hypothesized that the initiating and maintaining by Tbx3 and BMP4-mediated EMT signal pathway may be an important mechanism on regulating the directed differentiation of pacemaker cells. Our subject will to reveal the spatial and temporal expression of EMT signal during the differentiation of pacemaker cells of Tbx18+proepicardium in vivo lineage-tracing mice, Tbx18 gene knockout and conditional Snail gene knockout will reveal its influence on EMT and pacemaker cell differentiation; The horizontal and vertical interventions on EMT signal to reveal its impact on directional differentiation of pacemaker cells in vitro lineage-tracing mice. This topic is of great significance to reveal the mechanism of the directed differentiation of the functional pacemaker cells. This has significant meaning for elucidating the mechanism of the differentiation of pacemaker cells.

揭示心脏起搏细胞的来源及分化调控机制对生物起搏器的研究意义重大。Tbx18+心脏祖细胞（CPCs）是前体心外膜来源的祖细胞群，窦房结起搏细胞主要来源于该祖细胞，上皮间质转化(EMT)与前体心外膜祖细胞分化密切相关。但Tbx18+CPCs是否通过EMT信号调控起搏细胞定向分化，尚不明确。故提出假说：Tbx3及BMP4介导的EMT启动和维持可能是调控Tbx18+CPCs定向分化功能性起搏细胞的重要机制。本课题拟用Tbx18谱系示踪小鼠揭示Tbx18+前体心外膜体内起搏细胞分化过程中EMT信号的时空表达；用Tbx18基因敲除和条件性Snail基因敲除小鼠揭示其对EMT信号和起搏细胞分化的影响；用Tbx18体外示踪模型，横向和纵向干预EMT信号揭示其对起搏细胞体外定向分化的影响。以期阐明EMT信号在调控Tbx18+CPCs分化起搏细胞中的作用。本课题对揭示功能性起搏细胞定向分化的机制具有重要意义。

研究心脏起搏细胞的来源及分化机制对构建生物起搏器具有重要意义。本课题最初提出假说：EMT 信号的启动和维持可能是调控 Tbx18+前体心外膜祖细胞定向分化功能性起搏细胞的重要机制。为证实以上假说，本课题从以下三个方面进行了研究：（1）生理状态下观察前体心外膜向心脏起搏细胞定向分化过程中EMT 信号时空表达模式。（2）激活及阻断心脏起搏细胞分化相关上游因子，明确其对下游 EMT 信号分子、SAN 发育相关基因的影响。（3）体外 BMP4干预对 EMT信号及功能性起搏细胞分化的影响；阻断和激活 EMT 信号对 Tbx18+CPCs 定向诱导分化起搏细胞比率及起搏细胞功能的影响。主要研究成果及进展情况包括以下5部分：（1）建立Tbx18+CPCs谱系示踪模型，揭示父系与母系来源Cre双杂合示踪小鼠模型遗传特性具有差别；体内示踪生理状态下Tbx18+CPCs定向分化为心脏起搏细胞。（2）体外成功培养普通CPCs和Tbx18+CPCs，并研究其增殖、迁移、分化等生物特性。（3）揭示低氧、IGF信号对心外膜分化过程EMT机制的影响。（4）通过BMP4诱导Tbx18+CPCs向窦房结起搏细胞定向分化。（5）初步探讨人胚胎发育过程中CPCs的生物特性。综上所述，本课题初步验证假说：EMT 信号的启动和维持可能是调控 Tbx18+前体心外膜祖细胞定向分化功能性起搏细胞的重要机制。其具体调控机制尚需进一步研究。