Tet2及其介导的DNA去甲基化在iPS向造血细胞分化中的作用及机制研究

The hematopoietic cells that differentiated from iPS can be used as the best source for transplantation to treat the hematopoietic disorders. However, the low efficiency and poor quality seriously constraints its application and development. One important reason that inhibits this process is the abnormal DNA demethylation. It's recently found that Tet oxygenase (Tet1/Tet2/Tet3) can convert 5methylcytosine(5mC) to 5 hydroxymethylcytosine (5hmC), and thus involved in DNA demethylation, and Tet2 mutant recurrently appeared in multiple hematopoietic malignancies. Therefore, based on the works that we have already achieved, such as iPS induction and Tet1 can maintain the identity of iPS,.we will research the 5mC/5hmC levels on genome-scale, and the methylation status and expression of hematopoietic-related genes during the differentiation process from iPS to hematopoietic cells with immunofluorescence staining, bisulfite sequencing and gene knockout technology, and then we hope to uncover dynamic changes of the DNA demethylation during differentiation, whether Tet2 directly involved in the DNA demethylation and the effect of Tet2 on the differentiation potential of during differentiation. Thus, we aim to clarify the epigenetic regulation mechanism that how Tet2 and its mediated DNA demethylation influence the differentiation progress from iPS to hematopoietic lineages. Furthermore, this can uncover the relevance between DNA demthylation and the efficiency of hematopoietic differentiation, and provide abundant theoretical and experimental basis for improving the differentiation efficiency from iPS to hematopoietic lineages and promoting its early clinical application.

iPS细胞分化成的造血细胞可作为治疗造血疾病的最佳移植来源，但分化效率低下、质量不好严重制约着其发展和应用。DNA去甲基化异常是影响该过程的重要原因。最近发现Tet加氧酶介导的5甲基胞嘧啶向5羟甲基胞嘧啶转化参与DNA去甲基化,Tet2突变频发于多种造血肿瘤中。本项目在前期获得iPS细胞系和Tet1能维持iPS干性的工作基础上，拟运用免疫荧光染色、亚硫酸盐测序、基因敲除等技术，以iPS向造血分化过程为研究对象，检测全基因组5mC/5hmC水平、造血基因的表达及甲基化状态，从而明确分化过程中DNA甲基化动态变化、Tet2是否参与分化过程中甲基化调节以及Tet2是否影响造血分化潜能；旨在发现Tet2及其介导的DNA去甲基化对造血分化的影响，并初步阐明此作用的表观遗传调节机制。进而揭示DNA去甲基化与造血分化效率的关联性，为提高iPS向造血细胞分化效率、促进其早日达到临床提供丰富的理论和实验基础

DNA甲基化异常是体细胞重编程和干细胞定向分化过程中的重要表观遗传学障碍，但其调控机制仍不清楚。最近发现DNA去甲基化酶（TET家族）在干细胞多能性维持以及分中发挥重要作用。本项目以此切入点，通过经典的四因子组合成功建立了iPS细胞系。所建立的iPS细胞系呈现碱性磷酸酶阳性，表达OCT4、SOX2、NANOG以及SSEA1等干细胞标记分子，并且具有一定的发育潜能，体外可以形成拟胚体实验，体内可以形成畸胎瘤。在正常的iPS细胞基础上，通过Crispr/Cas9的方法建立了Tet2基因敲除的iPS细胞系。通过蛋白纯化系统体外纯化获得了TET2蛋白，纯化的TET2蛋白在体外具有加氧酶的活性。探索了TET2的辅因子α-酮戊二酸（α-KG）在重编程中的作用，发现α-KG对细胞生长具有双重作用，并且能够加速体细胞重编进程、提高重编程效率。此外，使用体外方法将iPS细胞分化形成了CD34+的造血祖细胞。本研究的结果增加了TET2及其辅因子通过影响表观遗传状态而调控体细胞重编程方面的研究，丰富了细胞代谢产物通过表观遗传调节而调控细胞增殖和干细胞命运决定的知识，为iPS的诱导以及定向分化提供了理论和实验依据。