發(fā)布時(shí)間：2018-06-07 10:22

  本文選題：Ltv1 + 核糖體疾病�。� 參考：《西南大學(xué)》2017年碩士論文

【摘要】：核糖體幾乎存在于所有細(xì)胞中,作為蛋白質(zhì)合成的場(chǎng)所,核糖體是一切細(xì)胞活動(dòng)的基礎(chǔ)。核糖體的合成是一個(gè)復(fù)雜而又相當(dāng)消耗能量的過程。在真核細(xì)胞中,核糖體的合成需要三種RNA聚合酶,四種核糖體RNA(rRNA),約70種小核仁RNA(snoRNA),82種核糖體蛋白和超過200種的非核糖體蛋白。遺傳突變導(dǎo)致核糖體合成過程紊亂會(huì)導(dǎo)致一系列的疾病,人們將這一類疾病稱為核糖體病。盡管所有核糖體病都有核糖體合成過程的異常,但是它們卻表現(xiàn)出不同的臨床癥狀,致病機(jī)制以及潛在的治療方法。核糖體病的癥狀常常包括消化器官與顱面軟骨缺陷,血細(xì)胞異常以及易發(fā)癌癥等。為了研究核糖體病的機(jī)制,人們建立了很多相關(guān)的動(dòng)物模型。斑馬魚作為一種研究胚胎早期發(fā)育的理想模式動(dòng)物,越來越多地被應(yīng)用于核糖體病機(jī)制的研究。研究者在斑馬魚中構(gòu)建了很多尚未在人類核糖體病中檢測(cè)到的核糖體相關(guān)基因的突變模型,為核糖體病的鑒定提供了新的參考基因同時(shí)也為核糖體病的機(jī)制的研究與治療方案的篩選提供了重要的研究載體。Ltv1是一種非核糖體蛋白,參與40s核糖體亞基的運(yùn)輸。在酵母,果蠅和人類細(xì)胞系中,ltv1缺陷會(huì)導(dǎo)致18s rRNA加工過程的異常。酵母中缺失ltv1會(huì)導(dǎo)致細(xì)胞的生長(zhǎng)受到抑制。在果蠅中敲除ltv1會(huì)導(dǎo)致果蠅幼蟲的發(fā)育阻滯并在幼蟲期死亡。這些研究都表明ltv1是一個(gè)功能保守的核糖體合成相關(guān)的基因。但是目前關(guān)于ltv1在脊椎動(dòng)物的發(fā)育過程中的功能的研究較少。我們利用CRISPR/Cas9技術(shù)在斑馬魚中敲除了ltv1,獲得了兩種突變類型ltv1Δ7和ltv1Δ14。ltv1~(-/-)突變體中的18s rRNA加工過程異常,這表明了ltv1在rRNA加工中功能的保守性。觀察發(fā)現(xiàn),5dpf時(shí)ltv1~(-/-)突變體表現(xiàn)出頭部稍小,心包水腫,魚鰾不能充氣,腸道不能回轉(zhuǎn)以及卵黃吸收不良的表型。由于很多核糖體病患者都表現(xiàn)出消化器官缺陷,顱面骨骼以及血液系統(tǒng)異常等癥狀,因此,我們著重觀察了ltv1~(-/-)突變體在這三個(gè)方面的表型。研究發(fā)現(xiàn),ltv1~(-/-)突變體的顱面軟骨中的麥克爾軟骨變小,顎方骨變彎并且角舌骨和5對(duì)角腮骨全部缺失。在消化器官中,ltv1~(-/-)突變體的肝臟,腸道和外分泌胰腺表現(xiàn)出明顯的發(fā)育不良。肝臟發(fā)育的起始與細(xì)胞分化并不受影響。在血液細(xì)胞中,ltv1~(-/-)突變體的永久造血中的各種血液細(xì)胞包括造血干細(xì)胞前體細(xì)胞(HSPC),紅細(xì)胞,巨噬細(xì)胞,嗜中性粒細(xì)胞和淋巴細(xì)胞都顯著減少,而原始造血中紅細(xì)胞前體和髓系細(xì)胞前體并不受影響。這些表型都與核糖體病的癥狀相似。進(jìn)一步分析表明ltv1~(-/-)突變體外分泌胰腺和HSPC的缺陷是由于外分泌胰腺前體細(xì)胞以及HSPC的增殖減少導(dǎo)致的,而凋亡水平在ltv1~(-/-)突變體中沒有明顯變化。很多核糖體病的動(dòng)物模型都報(bào)道了p53參與的機(jī)制,而在ltv1~(-/-)突變體中,p53的靶基因Δ113p53和p21表達(dá)明顯上升,但是下調(diào)p53卻不能挽救ltv1~(-/-)突變體的表型。綜上所述,我們構(gòu)建了ltv1~(-/-)的斑馬魚突變體,并驗(yàn)證了ltv1在18s rRNA加工中的保守作用,同時(shí)揭示了ltv1對(duì)于消化器官,血液以及顱面軟骨發(fā)育的重要性。進(jìn)一步的分析揭示了p53的靶基因Δ113p53和p21在ltv1~(-/-)突變體中上調(diào)但表型并不依賴于p53�？偠灾�,我們構(gòu)建了一個(gè)新的核糖體病斑馬魚模型,為核糖體病的鑒定提供了新的參考基因,并可用于研究核糖體病的分子生物學(xué)和細(xì)胞生物學(xué)機(jī)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R596

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 Yong Wang;Qinfang Zhu;Ling Huang;Yanqing Zhu;Jun Chen;Jinrong Peng;Li Jan Lo;;Interaction between Bms1 and Rcl1,two ribosome biogenesis factors,is evolutionally conserved in zebrafish and human[J];Journal of Genetics and Genomics;2016年07期

2 Jason Ear;Jessica Hsueh;Melinda Nguyen;QingHua Zhang;Victoria Sung;Rajesh Chopra;Kathleen M.Sakamoto;Shuo Lin;;A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure[J];Journal of Genetics and Genomics;2016年05期

3 ;Ribosome Biogenesis Factor Bmsl-like Is Essential for Liver Development in Zebrafish[J];遺傳學(xué)報(bào);2012年09期

，

本文編號(hào)：1990835