嘌呤能P2受体对人胚胎干细胞及其分化的心血管前体细胞自我更新和分化的调控作用及机制

So far, very little is known about the information in human early embryological development due to the ethic and technical limitation. Human embryonic stem cells (hESCs) have been verified to act as an ideal in vitro model for the study of human embryonic development. hESCs are long-term self-renewing cells that are able to differentiate into any cell type in the body. hESCs derived cardiovascular progenitor cells(CVPCs) have been shown to be a promising source for myocardial regeneration. Therefore, it is of great importance to explore the mechanisms of the maintenance of pluripotency and differentiation of hESCs. It has been suggested that the alteration of expression of purinergic P2 receptors during development indicates specific functions for progression of ESC differentiation and final phenotype specification. P2 receptors are classified into two subtypes (P2X and P2Y). Several endogenous activators are recognized by P2 receptors, among which adenosine triphosphate (ATP) is the most common extracellular signaling molecule. These receptors play key roles from the very beginning of life. They have been shown to play important roles in the early development of amphibians, birds and mammals. However, whether human embryos express P2 receptors is still unknown. Both P2X and P2Y receptors are able to modulate intracellular Ca2+ signals. Some important Ca2+ related proteins, located on cell membrane and sarcoplasmic reticulum, participate in maintaining Ca2+ homeostasis. Although there is evidence suggesting the presence of basic Ca2+ regulating pathways in mouse ESCs, it is still not demonstrated in hESCs. In addition, regulation of hESC self-renewal and the cell fate can be modulated by the coordinated actions of extrinsic regulators and signaling pathways, such as the Ca2+ signaling. Our latest results indicate that hESCs express P2 receptors at the molecular level and the intracellular Ca2+ signals of hESCs can be modulated by P2 receptors. Based on the above, we hypothesize that P2 receptors may be involved in hESC self-renewal and their differentiation. The aim of our project is the following: 1) to clarify the expression pattern of P2 receptors in hESCs and hESCs-derived CVPCs; 2) to examine if P2 receptors modulate hESC self-renewal; 3) to determine the role of P2 receptors in the differentiation of hESCs into CVPCs; 4) to explore the effect of P2 receptors on the intracellular Ca2+ signals and characterize the functional properties of calcium mobility in hESCs and hESCs-derived CVPCs. It is very important to promote these studies and the results may be helpful to understand the mechanisms of human early embryonic development.

人胚胎干细胞(hESCs)是早期胚胎发育理想的体外研究模型，hESCs向心血管前体细胞(CVPCs)的定向分化有助于获得充足的细胞用于移植治疗心脏疾病。胞膜嘌呤能P2受体(P2R)可调控动物胚胎发育，但P2R在人早期胚胎是否有表达及其功能调控作用不清楚。我们新近发现hESCs及分化的CVPCs表达P2R，且钙信号受P2R调控。因此我们推测P2R介导的钙信号可能在hESCs自我更新及向CVPCs分化中起重要作用。为验证此设想，本项目拟通过分子生物学、细胞生物学、功能学检测等手段探讨以下问题：1) P2R在hESCs及其分化的CVPCs的表达模式；2) P2R是否参与对hESCs自我更新的调节；3) P2R对hESCs向CVPCs分化过程的影响；4) P2R是否参与调控hESCs和CVPCs的钙活动及调控通路如何。这方面的研究有助于丰富对人胚胎早期发育调控的了解，具有重要的生物学意义。

人胚胎干细胞（hESCs）是早期胚胎发育理想的体外研究模型，hESCs向心血管前体细胞（CVPCs）的定向分化有助于获得充足的细胞用于移植治疗心脏疾病。胞膜嘌呤能P2受体（P2R）可调控动物胚胎发育，并能通过调控细胞内重要的第二信使钙离子相关信号通路影响一系列生理活动。但P2R在hESCs及其分化的CVPCs是否有表达及其对钙离子功能调控作用尚不清楚。本项目通过分子生物学、细胞生物学、功能学检测等多种手段明确了hESCs及分化的CVPCs表达不同亚型P2R，且P2R参与调控hESCs及CVPCs的胞内钙信号，而三磷酸肌醇（IP3）受体是介导P2R调控钙信号这一作用的主要内质网钙释放受体，而且不同亚型IP3受体与不同P2R亚型相偶联发挥作用；此外，我们还发现hESCs在分子水平上表达多种胞膜和内质网钙通道相关蛋白的基因，这些钙通道在hESCs钙稳态调控中发挥重要作用。具体研究结果如下：1) hESCs及其分化的CVPCs表达P2R；2) P2Y受体是介导hESCs和CVPCs中嘌呤能钙信号的主要P2受体亚型；3）P2Y1受体是介导hESCs而非CVPCs中嘌呤能钙活动的主要P2Y受体亚型；4）IP3受体亚型2（IP3R2）与P2Y1受体耦联介导hESCs中的钙信号；5）IP3受体亚型3（IP3R3）和IP3R2在P2受体介导CVPCs钙信号中发挥不同作用；6）胞膜和内质网钙通道/钙泵在hESCs钙信号稳态调控中具有重要作用。综上所述，本项目的研究发现将有助于丰富对人胚胎早期发育调控的了解，对揭示hESCs自我更新及向CVPCs分化过程中的钙信号调节通路具有重要的生物学意义。