PCGF家族蛋白在人红系祖细胞增殖和分化中的作用研究

红细胞输注是临床上应用最广泛的细胞治疗手段，来源主要依赖志愿者外周血捐献。供者不足、感染风险、稀有血型缺乏等仍是世界性的输血难题。过去几十年全世界科学家都致力于开发红细胞替代品用于输血，但均未成功。因此，如何在体外大量生产成熟红细胞已成为该领域的重大挑战和研究热点。我们前期研究建立了从人外周血单个核细胞大量扩增红系祖细胞并高效诱导分化为成熟红细胞的实验体系，首次发现外源表达PCGF4/BMI1基因可以促使红系祖细胞体外扩增高达一万亿倍，同时保留了进一步分化成熟的潜力。在此工作基础上，本项目将以红系祖细胞体外扩增和分化脱核为研究模型，采用慢病毒感染的方式进行人多梳环指蛋白家族（PCGF）基因的敲低和过表达，系统地探索PCGF家族其余成员在人红系祖细胞增殖分化中的作用，并进一步通过多组学分析探讨PCGF-PRC1介导的表观遗传调控机制，为体外制备大量成熟红细胞并应用于临床提供理论依据。

Transfusion of red blood cells (RBCs), which are mostly donated from healthy donors, is a standard and indispensable cellular therapy in current clinical practice. Due to limited donor availability, risks of pathogen contaminations and potential allo-immunization to donor RBCs even after ABO blood-type matching, ex vivo production of functional erythrocytes in a laboratory setting is highly desirable and has been pursued in the past decades. However, generating physiological numbers of transfusable cultured RBCs (cRBCs) ex-vivo remains challenging, due to our inability to either extensively expand primary RBC precursors (erythroblasts) or achieve efficient enucleation once erythroblasts have been expanded and induced to differentiation and maturation. In our previous study, we found that ectopic expression of the human PCGF/BMI1 gene confers extensive expansion of human erythroblasts, which can be derived readily from adult peripheral blood mononuclear cells of either healthy donors or sickle cell patients. These extensively expanded erythroblasts (E3) are able to proliferate exponentially (>1 trillion fold in 2 months) in a defined culture medium. Expanded E3 cells are karyotypically normal, and capable of terminal maturation with approximately 50% enucleation. Additionally, E3-derived cRBCs can circulate in a mouse model following transfusion similar to primary human RBCs. Based on these results, we will continue to investigate roles of polycomb group RING finger proteins on the proliferation and differentiation of human erythroblasts through lentivirus mediated gene knockdown and ovexpression. Furthermore, the second goal of this proposal is to explore the mechanism of PCGF-PRC1 related epigenetic regulation. This new project is highly innovative both conceptually and technologically. The success of this project will provide key evidence for generation of large-scale mature RBCs from extensively expanded erythroblasts for clinical applications.