BCAS2参与斑马鱼造血系统发育的分子机制研究

The vertebrate hematopoiesis signal network is very complex and needs precise control, whose imbalance may lead to severe dysplasia. The fine regulation of hematopoiesis is not yet completely clear. BCAS2 is a component of spliceosome. It may play important roles in the differentiation and development of eukaryotic cells. In our preliminary work, we have applyed TALEN technology in constructing a BCAS2 knockout zebrafish model, in which significantly decreased juvenile blood cell number was identified, suggesting that BCAS2 may play an important role in the development of hematopoietic system. Based on the preliminary work, observation and analysis of the hematopoietic system development in different periods of the wild type and BCAS2-/- zebrafish will be carried out. And we will study effects of BCAS2 mutation on Notch and/or cmyb/sdf1 pathway, through wich the hematopoiesis process may be affected. We will also study whether BCAS2 regulates the hematopoitic stem cell number by affecting their survival and proliferation. Futhermore, we will investigate the transcriptome of zebrafish at different stages to uncover novel hematopoitic signalling pathways. This project will explore new mechanisms of BCAS2 in eukaryotic cells and will benefit the fully understanding about regulation network in hematopoiesis.

脊椎动物的造血过程受复杂而精密的信号网络所调控，其失衡可能导致严重发育异常。BCAS2是剪接复合体的一个成份，可能在真核细胞分化与发育中扮演重要角色，但对其研究尚少。前期工作中，我们应用 TALEN 技术构建了BCAS2敲除的斑马鱼模型，发现纯合缺失的幼鱼造血干细胞的迁移和分化异常，提示BCAS2可能在造血系统发育中发挥重要作用。本课题将在前期工作基础上，研究并确定BCAS2-/-斑马鱼造血干细胞发育和分化缺陷的具体阶段；研究BCAS2基因敲除是否通过Notch、cmyb/sdf1a信号通路影响造血干细胞发育或迁移, 是否通过调控细胞存活与增殖影响造血干细胞数量；通过转录组研究探索BCAS2影响造血干细胞发育新的分子信号网络并对其进行验证。本项目对于深入了解BCAS2在细胞分化和组织发育中的功能地位，和发现造血调控网络的新通路，均有重要意义。

造血是各种血细胞发育、分化、成熟的复杂有序的动态过程，受多种因子严密调控；我们前期工作建立了稳定遗传的斑马鱼bcas2基因敲除模型，首次发现剪接因子BCAS2参与斑马鱼造血过程，提示脊椎动物体内可能存在新的造血干细胞发育和分化的调控通路。本课题对BCAS2缺陷影响斑马鱼造血的具体阶段及其分子调控机制进行了深入研究。.我们利用bcas2-/-斑马鱼模型，发现在斑马鱼中敲除bcas2不影响初级造血以及红髓系祖细胞的正常形成，但发育至3-4dpf时，bcas2-/-斑马鱼中造血干细胞数目明显减少，且在5dpfbcas2-/-斑马鱼尾部造血组织中仅残留少量的造血干细胞，表明bcas2-/-斑马鱼次级造血缺陷；我们通过TUNEL方法发现细胞凋亡可能是造成造血干细胞减少的重要原因；通过RT-PCR与western blot方法检测到bcas2-/-斑马鱼中p53及其下游基因p21、MDM2、PUMA、NOXA与 BAX的表达显著上调，且过表达p53可部分回补造血干细胞数目，表明bcas2-/-斑马鱼造血干细胞凋亡是p53依赖的；进一步地，我们通过半定量PCR和RT-PCR检测了Mdm4-FL和Mdm4-S在野生型及bcas2-/-斑马鱼中的表达情况，并利用相关的回补实验，发现bcas2缺失所导致的Mdm4剪接异常，是p53活化与细胞凋亡的主要原因，提出了bcas2-/-斑马鱼中造血缺陷的分子调控通路。.本课题在首次发现剪接因子BCAS2影响造血发育的基础上，明确了其调控造血的相关信号通路和分子机制，对于揭示剪接因子在发育生物学的功能和探索造血调控的新通路具有重要意义