PDGF-C在调控胚胎干细胞自我更新和多能性中的作用及机制研究

Embryonic stem cells (ESCs) are capable of self-renewal and pluripotency, therefore have great value for both basic research and clinical application. However, the role and mechanism of key growth factors in regualting ESCs are still unclear, which hinders ESCs large scale application. As a newer member of platelet-derived growth factor (PDGF) family, PDGF-C has various functions including the regulation of cell division and angiogenesis, but its role in ESCs remains unknown. Our preliminary data show that the expression of PDGF-C decreases during mouse ESCs (mESCs) differentiation. Moreover, addition of PDGF-C protein significantly enhances mESCs proliferation, and promotes the expression of pluripotency marker while represses mesoderm marker. In this project, we plan to study the functions of PDGF-C in regulating mESCs self-renwal and pluirpotency using overexpression and RNAi systems, combined with cellular and molecular biology, as well as immunohistochemical staining methods. Then the impact of PDGF-C on early embryo development will be examined by PDGF-C knockout mice model. Finally, we will explore its downstream targets and signaling pathways by receptor phosphorylation assay and microarrays. This study will not only reveal the functions and mechanisms of PDGF-C in ESCs regulation, but also provide new methods and solutions for stem cells clinical transformation in the future.

胚胎干细胞（ESCs）具有自我更新和多能性的特性，对基础研究与临床应用均有重要价值。但调控ESCs的关键生长因子及其作用机制仍未完全明了，限制着ESCs规模化应用。血小板源性生长因子C (PDGF-C) 是PDGF家族较新的成员，具有促细胞分裂与血管形成等功能，但其在ESCs中作用未知。申请人前期研究发现PDGF-C在小鼠ESCs分化后表达下调；添加PDGF-C蛋白可显著促进小鼠ESCs细胞增殖、提高多能性因子和抑制中胚层标志物的表达，提示其在ESCs中具有重要调控作用。本项目拟利用过表达及RNAi技术，结合细胞与分子生物学、免疫组化等方法研究PDGF-C在小鼠ESCs自我更新及多能性中的作用；利用PDGF-C敲除小鼠模型明确其对早期胚胎发育的影响；通过受体磷酸化、芯片技术等确定其下游调控靶点与通路。本研究将阐明PDGF-C调控ESCs的作用及机制，为干细胞临床转化提供新的思路和方法。

胚胎干细胞（ESCs）具有自我更新和多能性两大特性，但目前对于调控其的关键生长因子及其作用机制仍未明了，限制了ESCs体外大量扩增的效率及安全性。血小板源性生长因子PDGF-C具有促血管新生、细胞增殖等功能，但其ESCs及小鼠胚胎发育中的作用仍未知。我们的前期数据显示PDGF-C在小鼠ESCs分化后表达下调；重组PDGF-C可显著促进小鼠ESCs细胞增殖、提高多能性因子和抑制中胚层标志物的表达，提示其在ESCs中具重要调控作用。本项目主要采取多种分子、蛋白、细胞等技术，结合体外及体内实验，解析PDGF-C对小鼠ESCs的干性调控作用及机制，以及对小鼠胚胎发育的影响。在细胞层面及畸胎瘤实验，我们发现PDGF-C具有促ESCs增殖、自我更新及多能性的作用，通过RNA microarray发现PDGF-C的敲低会引起分化相关的基因上调、干性相关的基因下调。另外，我们也发现PDGF-C在ESCs中可激活PDGFRa和b两个受体的表达从而影响细胞的干性。在体内，我们发现PDGF-C敲除会导致小鼠出生后严重致死，而注射PDGF-C蛋白可部分挽救胚胎体重, PDGF-C敲除也会下调早期胚胎中Nanog的表达。最后，我们进行单细胞测序实验分析，发现PDGF-C敲除后会导致分化相关的基因富集，并且在细胞周期、细胞亚群上与野生型细胞具有明显区别。本项目阐明了PDGF-C对ESCs及胚胎的调控作用，并明确其作用的分子机制，为优化ESCs体外培养、胚胎发育效率的提高带来新的思路。