下调rpl15基因的表达抑制斑马鱼早期造血的发育  

Down-regulation of rpl15 gene expression inhibits development of early hematopoiesis in zebrafish

作　　者：周唯君 周栋珍 彭涛 杨懿琛 周艳华 李化 舒莉萍 何志旭 ZHOU Weijun;ZHOU Dongzhen;PENG Tao;YANG Yichen;ZHOU Yanhua;LI Hua;SHU Liping;HE Zhixu(Department of Immunology,School of Basic Medicine,National&Guizhou Joint Engineering Laboratory for Cell Engineering and Biomedicine Technique,Guizhou Provincial Key Laboratory of Regenerative Medicine,Guizhou Medical University,Guiyang,Guizhou Province,550004;Key Laboratory of Adult Stem Cell Translational Research,Chinese Academy of Medical Sciences,Guiyang,Guizhou Province,550004;Department of Pediatrics,the Affiliated Hospital of Zunyi Medical University,Zunyi,Guizhou Province,563000,China)

机构地区：[1]贵州医科大学细胞工程生物医药技术国家地方联合工程实验室,贵州省再生医学重点实验室,基础医学院免疫学教研室,贵阳550004 [2]中国医学科学院成体干细胞转化研究重点实验室,贵阳550004 [3]遵义医科大学附属医院儿科学教研室,贵州遵义563000

出　　处：《第三军医大学学报》2021年第17期1611-1618,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金地区基金项目(31860325);贵州省科技计划项目(黔科合基础〔2020〕1Y092)。

摘　　要：目的探究核糖体蛋白L15(ribosomal protein l15,rpl15)基因表达下调对斑马鱼早期造血发育的影响。方法设计并合成rpl15吗啉代反义寡核苷酸(morphilino antisense oligo,MO),通过显微注射到单细胞期斑马鱼(0~0.75 hpf)的受精卵中来下调rpl15的表达,通过qRT-PCR和Western blot技术验证下调rpl15基因表达的有效性。使用体式显微镜分别观察注射rpl15 MO(MO)组与野生型(WT)组的发育情况。O-dianisidine染色检测血红蛋白表达的情况,qRT-PCR检测红系特异性转录因子hbae3、hbbe1的表达变化。全胚原位杂交和qRT-PCR检测红系特异性转录因子gata1、髓系转录因子pu.1、粒系转录因子mpo、定向造血标志转录因子runx1和c-myb、原始祖细胞造血标志物scl和lmo2等早期造血发育过程的关键转录因子的表达变化。TUNEL实验检测造血组细胞的凋亡变化。结果显微注射rpl15 MO能显著下调斑马鱼rpl15的表达,与WT组相比,MO组表现出体节弯曲、发育迟缓、心包水肿等异常表型。MO组血红蛋白合成量与hbae3、hbbe1的表达量相较于WT组明显减少(P<0.05)。原位杂交及qRT-PCR结果表明,gata1的表达明显减少(P<0.05),pu.1、mpo、runx1、c-myb的表达无明显变化,scl的表达明显减少(P<0.01),lmo2的表达无明显变化。TUNEL实验显示造血祖细胞的凋亡无明显变化。结论下调rpl15的表达抑制了斑马鱼早期的造血发育,并可能与gata1和scl表达明显降低有关。Objective To investigate the effect of down-regulation of ribosomal protein l15(rpl15)gene on early hematopoietic development in zebrafish.Methods The rpl15 morphilino antisense oligo(MO)was designed and synthesized to down-regulate the expression of rpl15 by microinjection into embryo of zebrafish(0~0.75 hpf)at the unicellular stage.qRT-PCR and Western blotting were used to verify the effectiveness of down-regulating rpl15 gene.The development of the injected(MO)and wild-type(WT)groups were observed separately using a somatic microscope.O-dianisidine staining was used to detect the expression of hemoglobin.qRT-PCR was used to detect the changes in the expression of the red lineage-specific transcription factors hbae3 and hbbe1.Whole embryo in situ hybridization and qRT-PCR were performed to detect the changes in the expression of key transcription factors of early hematopoietic development such as red lineage-specific transcription factor gata1,myeloid transcription factor pu.1,granulocyte marker mpo,targeted he matopoietic marker transcription factors runx1 and c-myb,and primitive group cell hematopoietic markers scl and lmo2.TUNEL assay was used to detect the changes of apoptosis in hematopoietic group.Results Microinjection of rpl15 MO successfully down-regulated the expression of rpl15 in zebrafish,and the MO-injected group exhibited abnormal phenotypes such as body segment curvature,developmental delay,and pericardial edema when compared with the control group.Hemoglobin synthesis was significantly reduced in the MO group than the control group.The results of in situ hybridization indicated that the expression of gata1 was significantly reduced(P<0.05),but the mRNA levels of pu.1,mpo,runx1,c-myb and lmo2 were not affected,while that of scl was obviously reduced(P<0.05).TUNEL assay showed no significant change in apoptosis of hematopoietic progenitor cells.Conclusion Down-regulation of rpl15 inhibits the early hematopoietic development of zebrafish,and it may be related to the significantly reduced expressio

关 键 词：rpl15 造血发育 斑马鱼 吗啉代反义寡核苷酸 

分 类 号：R-332[医药卫生] R331.2