PRTG蛋白调控ISL1在人胚胎干细胞向心肌分化过程中的作用机制研究

Congenital heart disease (CHD), caused by abnormal development of heart, is one of the most common congenital malformations. Transcription factor ISL1, a marker of second heart field cardiac progenitor cells, plays a key role in early heart development. However, the mechanisms remain unclear. Transmembrane protein protogenin (PRTG), a member of immunoglobulin superfamily, has multiple roles during embryogenesis, including neural and mesodermal development. In our preliminary work, PRTG was highly expressed in human embryonic heart and human embryonic stem cell-derived cardiac progenitor cells. The expression of PRTG showed a significant positive correlation with the expression of ISL1. Furthermore, one mutation of PRTG was found in our CHD patients. Thus we hypothesize that PRTG plays an important role in human cardiac development by regulating the expression of ISL1. In this proposed study, we will use the PRTG-knockout/overexpression embryonic stem cell lines to investigate the role of PRTG in cardiac differentiation. Next, we will illustrate the PRTG is involved in cardiovascular differentiation through up-regulating ISL1. Additionally, we will explore the mechanism of PRTG regulating ISL1 expression by Co-IP and CHIP assays. This study may provide a feasibility for illustration the function of PRTG on ISL1 during the heart development, which is expected to intensify our understanding of regulation mechanisms underlying the congenital heart disease.

先天性心脏病由胚胎时期心脏发育异常所致。心脏发育早期的核心转录因子ISL1，是第二生心区心脏祖细胞的分子标志，但其作用机制尚未完全阐明。跨膜蛋白PRTG是免疫球蛋白家族的一员，在神经和中胚层分化发育中具有重要作用。本课题组前期研究发现PRTG在早期胚胎心脏和人胚胎干细胞来源的心脏祖细胞中高表达，并与ISL1表达时间趋势呈高度一致性，先心病患儿中发现1例PRTG基因的有害突变。据此我们推测PRTG通过上调ISL1的表达参与心脏祖细胞的分化及心脏发育。本项目拟深入研究：1.通过PRTG敲除和过表达胚胎干细胞系的建立，探讨PRTG在人胚胎干细胞心血管系统定向分化过程中的作用。2.明确PRTG是否通过ISL1参与调控心血管的分化。3.采用Co-IP和CHIP实验，阐明PRTG是通过何种方式调控ISL1的表达。本研究将明确PRTG对ISL1的调控作用，为完善心脏发育中的调控网络提供理论依据。

先天性心脏病（Congenital heart defects，CHD）简称先心病，是一种最常见的出生缺陷疾病。跨膜蛋白基因突变或缺失会导致心脏发育异常。跨膜蛋白protogenin(PRTG)在胚胎早期发育中具有多种重要功能，阻止早期神经元的分化，促进近轴中胚层向体节的发育。鸡胚早期发育中，PRTG在心脏部位高表达。.为研究PRTG在心脏发育中的作用，我们收集了早期人类胚胎组织（11s-15s），并采用干细胞向心肌细胞定向分化的模型进行探讨。.本课题首次发现PRTG在人类胚胎心脏发育重要时期及心脏中胚层、心脏祖细胞时期高表达。通过Crispr/cas9技术敲除PRTG后，干细胞多能性不受影响，而心肌分化效率明显下降。通过RNA-seq测序发现PRTG敲除后，Hand1的mRNA水平升高，而蛋白表达明显下降。共聚焦结果显示PRTG与Hand1在细胞核内存在共定位。上述结果提示PRTG通过调控Hand1表达影响干细胞向心肌细胞分化效率，说明PRTG参与心脏发育的调控网络，为先心病的致病机理提出新的方向，为先心病的防控提供新的思路。