【作者】 张恒；

【导师】 贾海波；

【作者基本信息】 华中科技大学 ， 遗传学， 2020， 硕士

【摘要】 核糖体蛋白是一种十分普遍的与RNA结合的蛋白质,核糖体蛋白不仅可以与r RNA结合参与蛋白质的合成,同时还通过核糖体以外的功能参与调控细胞增殖和细胞凋亡。当核糖体蛋白的表达出现问题时,会导致类似于贫血,发育不良与癌症等比较严重的疾病。再生性障碍贫血（Diamond-Blackfan Anemia,DBA）是最早发现的与核糖体蛋白相关的疾病。DBA是一种非常罕见的骨髓异常综合征,具体特征表现为血液中的红细胞发育不全,先天畸形以及更高的患癌症率。目前已经在病人中已经检测到大约19种核糖体蛋白基因的突变。虽然已有大量的研究,但是关于DBA的致病机制目前仍不清楚。核糖体蛋白S24（RPS24）基因最开始是在DBA病人中发现的,在大约2%的DBA病人中检测到了rps24和rps17突变。人类的rps24基因包含6个外显子,编码着40S核糖体亚基的一个成分蛋白。基因敲低实验部分降低了靶标基因的蛋白水平。基因敲除后,靶标基因的蛋白完全丧失功能。两种实验中靶标基因的表达都存在缺陷,所以我们可以借鉴敲低实验的结果。前期已有研究已经在斑马鱼中建立了通过rps24 morpholino（MO）敲低导致的DBA模型。在rps24敲低的实验中,rps24的正常表达对于斑马鱼初级造血和次级造血发育都是必须的。为了进一步探究rps24突变对与斑马鱼造血可稳定遗传的影响,我们利用CRISPR-Cas9技术对斑马鱼rps24进行基因敲除,利用可稳定遗传的突变体来进一步探究rps24对斑马鱼早期造血的影响。通过CRISPR-Cas9我们成功的获得了斑马鱼rps24突变体斑马鱼。通过scl与gata1的整体原位杂交实验,我们发现rps24缺失不影响斑马鱼初级造血过程中的血管母细胞与初级造血红系细胞的发育。利用受精后36小时（36 hour post-fertilization,36 hpf）的胚胎,用runx1探针进行原位杂交,发现在36 hpf时,rps24^-/-斑马鱼中造血干细胞没有明显变化,但是在48 hpf时,rps24^-/-斑马鱼中的造血干细胞出现了减少。根据我们初步的表型研究推测rps24不影响斑马鱼内皮造血转化（endothelial-to-hematopoietic transition,EHT）过程,但影响造血干细胞的增殖和凋亡。邻联茴香胺染色和β-globin原位杂交实验结果表明rps24对斑马鱼红系发育是必须的,同时斑马鱼髓系细胞也出现了减少,但是在rps24^-/-斑马鱼中淋系的发育没有明显的变化。更多还原

【Abstract】 Ribosomal protein is a very ubiquitous RNA binding protein.Ribosomal protein can not only combine with r RNA to participate protein synthesis but also regulate cell proliferation and apoptosis in vivo through extra-ribosomal function.Therefore there will be serious diseases such as anemia,dysplasia and cancer when the expression of ribosomal protein become abnormal.Diamond-Blackfan anemia（DBA）is the first disease found to be associated with ribosomal protein.DBA is a rare bone marrow failure syndrome,which is characterized by hypoplastic erythrocytes,congenital malformations and more prone to cancer.19 genes have been found to be related with DBA in patients by now.Although there are lots of researches,it is still unclear about the pathogenesis of DBA.Ribosomal protein S24 gene was firstly found in patients with Diamond-Blackfan anemia（DBA）because of its mutation.Rps24 and rps17 mutation was found in about 2%DBA patients.The human rps24 gene which is consisted of 6 exons encodes a ribosomal protein that is a component protein of ribosomal 40S subunits.In knock down experiments,the target gene’s protein level reduces partly.In knock out experiments,the target gene’s protein loses its function.There are deficiencies of the target gene’s xepression in the two kinds of experiments.So we can refer to the results of knock down experiments.Rps24 knock down experiments through rps24 morpholino were carried out in zebrafish and corresponding DBA model was established before.In the rps24 knock down model of DBA,the normal expression of rps24 was found to be necessary in the primitive and definitive hematopoiesis of zebrafish.In order to further explore the heritable effect of rps24 on hematopoiesis of zebrafish,we knocked out the rps24 gene of zebrafish through CRISPR-Cas9 and made use of the stable heritable mutant zebrafish to further investigate the effect of rps24 on the early hematopoiesis of zebrafish.Through CRISPR-Cas9,the rps24 mutant zebrafish was successfully constructed.Through in situ hybridization experiment of scl and gata1,it was obvious that the development of angioblasts and primitive erythrocytes during primitive hematopoiesis was not affected when rps24 was knocked out.The results of in situ hybridization of runx1 and cmyb showed that the blood stem cells in rps24^-/-zebrafish were unchanged at36 hpf（hour after fertilization）but reduced at 48 hpf when compared to wild type zebrafish.We infer that rps24 don’t influence the endothelial-to-hematopoietic transition but influence blood stem cell’s proliferation and apoptosis.Results of O-Dianisidine and in situ hybridization ofβ-globin and mpo showed that rps24 was necessary for erythropoiesis and myelogenesis.However results of in situ hybridization of rag1 showed that the lymphocytopoiesis was unaffcted in rps24^-/-zebrafish when compared to wild type zebrafish.更多还原