核糖体生成因子Ltv1在斑马鱼中的功能研究

发布时间：2018-06-07 10:22

本文选题：Ltv1 + 核糖体疾病　；参考：《西南大学》2017年硕士论文

【摘要】：核糖体几乎存在于所有细胞中,作为蛋白质合成的场所,核糖体是一切细胞活动的基础。核糖体的合成是一个复杂而又相当消耗能量的过程。在真核细胞中,核糖体的合成需要三种RNA聚合酶,四种核糖体RNA(rRNA),约70种小核仁RNA(snoRNA),82种核糖体蛋白和超过200种的非核糖体蛋白。遗传突变导致核糖体合成过程紊乱会导致一系列的疾病,人们将这一类疾病称为核糖体病。尽管所有核糖体病都有核糖体合成过程的异常,但是它们却表现出不同的临床症状,致病机制以及潜在的治疗方法。核糖体病的症状常常包括消化器官与颅面软骨缺陷,血细胞异常以及易发癌症等。为了研究核糖体病的机制,人们建立了很多相关的动物模型。斑马鱼作为一种研究胚胎早期发育的理想模式动物,越来越多地被应用于核糖体病机制的研究。研究者在斑马鱼中构建了很多尚未在人类核糖体病中检测到的核糖体相关基因的突变模型,为核糖体病的鉴定提供了新的参考基因同时也为核糖体病的机制的研究与治疗方案的筛选提供了重要的研究载体。Ltv1是一种非核糖体蛋白,参与40s核糖体亚基的运输。在酵母,果蝇和人类细胞系中,ltv1缺陷会导致18s rRNA加工过程的异常。酵母中缺失ltv1会导致细胞的生长受到抑制。在果蝇中敲除ltv1会导致果蝇幼虫的发育阻滞并在幼虫期死亡。这些研究都表明ltv1是一个功能保守的核糖体合成相关的基因。但是目前关于ltv1在脊椎动物的发育过程中的功能的研究较少。我们利用CRISPR/Cas9技术在斑马鱼中敲除了ltv1,获得了两种突变类型ltv1Δ7和ltv1Δ14。ltv1~(-/-)突变体中的18s rRNA加工过程异常,这表明了ltv1在rRNA加工中功能的保守性。观察发现,5dpf时ltv1~(-/-)突变体表现出头部稍小,心包水肿,鱼鳔不能充气,肠道不能回转以及卵黄吸收不良的表型。由于很多核糖体病患者都表现出消化器官缺陷,颅面骨骼以及血液系统异常等症状,因此,我们着重观察了ltv1~(-/-)突变体在这三个方面的表型。研究发现,ltv1~(-/-)突变体的颅面软骨中的麦克尔软骨变小,颚方骨变弯并且角舌骨和5对角腮骨全部缺失。在消化器官中,ltv1~(-/-)突变体的肝脏,肠道和外分泌胰腺表现出明显的发育不良。肝脏发育的起始与细胞分化并不受影响。在血液细胞中,ltv1~(-/-)突变体的永久造血中的各种血液细胞包括造血干细胞前体细胞(HSPC),红细胞,巨噬细胞,嗜中性粒细胞和淋巴细胞都显著减少,而原始造血中红细胞前体和髓系细胞前体并不受影响。这些表型都与核糖体病的症状相似。进一步分析表明ltv1~(-/-)突变体外分泌胰腺和HSPC的缺陷是由于外分泌胰腺前体细胞以及HSPC的增殖减少导致的,而凋亡水平在ltv1~(-/-)突变体中没有明显变化。很多核糖体病的动物模型都报道了p53参与的机制,而在ltv1~(-/-)突变体中,p53的靶基因Δ113p53和p21表达明显上升,但是下调p53却不能挽救ltv1~(-/-)突变体的表型。综上所述,我们构建了ltv1~(-/-)的斑马鱼突变体,并验证了ltv1在18s rRNA加工中的保守作用,同时揭示了ltv1对于消化器官,血液以及颅面软骨发育的重要性。进一步的分析揭示了p53的靶基因Δ113p53和p21在ltv1~(-/-)突变体中上调但表型并不依赖于p53。总而言之,我们构建了一个新的核糖体病斑马鱼模型,为核糖体病的鉴定提供了新的参考基因,并可用于研究核糖体病的分子生物学和细胞生物学机制。
[Abstract]:Ribosomes are almost in all cells. As a place for protein synthesis, ribosomes are the basis of all cell activities. The synthesis of ribosomes is a complex and highly energy consuming process. In eukaryotic cells, the synthesis of ribosomes requires three kinds of RNA polymerase, four kinds of ribosome RNA (rRNA), about 70 small nucleoli RNA (snoRNA), and 82 species. Ribosomal proteins and more than 200 non ribosome proteins. Genetic mutations cause disorder in the process of ribosome synthesis resulting in a series of diseases. People call this disease ribosomal disease. Although all ribosomes have abnormal ribosome synthesis, they show different clinical symptoms, pathogenic mechanisms and potential. Treatment. The symptoms of ribosomal disease often include defects in the digestive organs and craniofacial cartilage, abnormal blood cells, and prone to cancer. In order to study the mechanism of ribosomal disease, many related animal models have been established. Zebrafish is more and more used in ribose as an ideal model animal to study the early development of embryos. Research on the mechanism of somatic disease. Researchers have constructed a number of mutant models of ribosome related genes that have not been detected in human ribosomal disease in zebrafish, providing a new reference gene for the identification of ribosomal disease and providing an important research carrier,.Ltv1, for the screening of mechanisms for ribosomal disease. Non ribosome proteins involved in the transport of 40s ribosome subunits. In yeast, Drosophila and human cell lines, LTV1 defects can lead to abnormal processes in the processing of 18S rRNA. The absence of LTV1 in yeast can cause cell growth to be inhibited. The knockout of LTV1 in the Drosophila may cause the growth block of Drosophila larva and die in the larval stage. All these studies table LTV1 is a functional conserved ribosome synthesis related gene. But there are few studies on the function of LTV1 in the development of vertebrates. We use CRISPR/Cas9 technology to knock out LTV1 in zebrafish, and obtain the abnormal 18S rRNA processing in two mutant types, LTV1 delta 7 and LTV1 Delta 14.ltv1~ (- / -) mutants. This shows the conservatism of LTV1's function in rRNA processing. It was observed that the ltv1~ (- / -) mutant of 5dpf showed a slightly smaller head, pericardial edema, no inflatable swim bladder, no gyration of the intestines, and a poor yolk phenotype. Many patients with ribosomal disease showed signs of organ defects, craniofacial bones, and blood system abnormalities. Therefore, we emphatically observed the phenotype of the ltv1~ (- / -) mutant in these three aspects. The study found that the Meckel cartilage in the craniofacial cartilage of the ltv1~ (- / -) mutant was smaller, the jaw bone became curved and the hornohyoid and 5 diagonal gills were all missing. In the digestive organs, the ltv1~ (- / -) mutant of the liver, the intestine and the exocrine pancreas were demonstrated. Significant dysplasia. The initiation and differentiation of the liver development is not affected. In the blood cells, the various blood cells in the ltv1~ (- / -) mutant's permanent hematopoiesis include hematopoietic stem cell precursor cells (HSPC), red blood cells, macrophages, neutrophils and lymphatic cells, and the original hematopoiesis erythrocyte precursor and pulp The cell precursors were not affected. These phenotypes were similar to the symptoms of ribosomal disease. Further analysis showed that the defects of the ltv1~ (- / -) mutation in the pancreas and the HSPC were caused by the proliferation of the exocrine pancreatic precursor cells and the proliferation of HSPC, and the apoptosis level was not significantly changed in the ltv1~ (- / -) mutant. Animal models all reported the mechanism of p53 participation, and in the ltv1~ (- / -) mutant, the expression of p53 target gene Delta 113p53 and p21 increased obviously, but the down-regulation of p53 could not save the phenotype of ltv1~ (- / -) mutants. In summary, we constructed a ltv1~ (- / -) zebrafish mutant, and verified the conservative effect of LTV1 in 18S rRNA processing. The importance of LTV1 to the development of digestive organs, blood and craniofacial cartilage was revealed. Further analysis revealed that the p53 target gene, Delta 113p53 and p21 were up regulated in the ltv1~ (- / -) mutant, but the phenotype was not dependent on p53.. We constructed a new ribosome mackfish model for the identification of ribosomal disease. Reference genes can be used to study the molecular biology and cell biology mechanism of ribosomal diseases.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R596

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