RNA结合蛋白Rbm38在小鼠胚胎造血发育中的功能研究

Functional study of RNA-binding protein Rbm38in mouse embryonic he⁃matopoietic development

目的:探究RNA结合蛋白Rbm38在小鼠造血干细胞(hematopoietic stem cell,HSC)发育中的作用。方法:分析造血干细胞发育过程中连续群体的单细胞转录组数据,筛选生血内皮细胞(hemogenic endothelial cell,HEC)和/或造血干细胞前体(hematopoietic stem cell precursor,pre-HSC)阶段表达上调的RNA结合蛋白分子(RNA binding protein,RBP);利用CRISPR/Cas9技术构建基因敲除小鼠模型,将杂合型小鼠交配获得实验用胚胎,按照小鼠胚胎基因型不同将实验分为实验组和对照组,实验组为Rbm38^(-/-)基因型胚胎,对照组为Rbm38^(+/+)和Rbm38^(+/-)基因型胚胎;取材胚胎期11.5 d(embryonic day 11.5,E11.5)孕鼠胚胎的卵黄囊(yolk sac,YS),行体外造血细胞集落形成实验(colony forming unit-culture,CFU-C)检测主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区和卵黄囊生成造血祖细胞(hematopoietic progenitor cell,HPC)的能力,行小鼠胚胎AGM区移植实验评价AGM区产生功能性造血干细胞的能力。结果:(1)Rbm38在动脉内皮细胞(arterial endothelial cell,AEC)向生血内皮细胞和Ⅰ型造血干细胞前体(type 1 pre-hematopoietic stem cell,T1 pre-HSC)的转化过程中表达明显上调,且在后续造血干细胞成熟的过程中维持高水平表达;(2)实验组和对照组E11.5小鼠胚胎AGM区和卵黄囊生成造血祖细胞数量以及其分化功能无统计学差异(P>0.05);(3)实验组和对照组移植后外周血嵌合率无统计学差异(P>0.05)。结论:(1)多种RNA结合蛋白分子,如Rbm38,在小鼠生血内皮细胞与Ⅰ型造血干细胞前体阶段特异性高表达;(2)敲除Rbm38不影响小鼠胚胎AGM区和卵黄囊生成造血祖细胞的能力;(3)敲除Rbm38不影响小鼠胚胎AGM区产生功能性的造血干细胞。

AIM:To investigate the role of RNA-binding protein Rbm38 during mouse hematopoietic stem cell development.METHODS:Single-cell transcriptomic data of continuous developmental hematopoietic stem cell-related populations was analyzed to screen for RNA-binding protein molecules that are upregulated at the hemogenic endothelial cell and/or hematopoietic stem cell precursor stages.The Rbm38 knockout mice were constructed by CRISPR/Cas9 technology.The heterozygous mice were bred to obtain experimental embryos.The embryos were divided into experimental group(Rbm38^(-/-))and control group(Rbm38^(+/+)and Rbm38^(+/-))according to their genotypes.The ability of hematopoietic progenitor cell generation in aorta-gonad-mesonephrosregion(AGM)and yolk sac was examined by in vitro colony-forming units-culture assay with yolk sacs from E11.5 embryos.Furthermore,the ability of functional hematopoietic stem cell generation in the AGM region was examined by AGM region direct transplantation assay in E11.5 embryos.RESULTS:(1)The expression of RNA-binding protein Rbm38 was significantly up-regulated during the transition from arterial endothelial cell to hemogenic endothelial cell and type 1 hematopoietic stem cell precursors,and maintained at a high level during subsequent hematopoietic stem cell maturation.(2)There was no statistical difference in the numbers of hematopoietic progenitor cell and the differentiation function of hematopoietic progenitor cell generated in the E11.5 AGM region or yolk sac between the experimental group and the control group(P>0.05).(3)There was no statistical difference in the peripheral blood chimerism between the experimental group and the control group after transplantation(P>0.05).CONCLU⁃SION:A variety of RNA-binding protein molecules,such as Rbm38,are specifically highly expressed in mouse hemogenic endothelial cell and type 1 hematopoietic stem cell precursor stages.Knockout of Rbm38 does not affect the generation of hematopoietic progenitor cell in AGM region or yolk sac of mouse embryos.Knockout of Rbm38 does not affect the generation of functional hematopoietic stem cell in the AGM region of mouse embryos.