本文關(guān)鍵詞：人類卟啉癥及貧血的斑馬魚疾病模型的建立與解析　出處：《蘇州大學(xué)》2015年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血紅素 卟啉癥 TALEN CRISPR-Cas9 alad cpox hmbsa hmbsb 深度測序 生物鐘 per1a

【摘要】：血紅素(heme)是生物體內(nèi)一種重要的分子,參與組成血紅蛋白、肌紅蛋白、細(xì)胞色素P450、維生素B1等。血紅素的合成由8步酶催化的級聯(lián)反應(yīng)組成,相應(yīng)的催化酶分別由對應(yīng)的基因編碼,包括ALAS1和ALAS2、ALAD、HMBS、UROS、UROD、CPOX、PPOX、FECH。血紅素合成過程中的酶的功能的缺陷導(dǎo)致一系列被稱為卟啉癥(porphyria)的綜合癥。卟啉癥的臨床癥狀復(fù)雜,目前的診斷和治療手段相對有限。斑馬魚作為一種模式生物,在人類疾病的研究中有著廣泛的應(yīng)用,其相應(yīng)基因缺陷的突變體能夠作為人類卟啉癥的疾病模型。已有報(bào)道斑馬魚alas2,urod,ppox,fech基因缺陷的突變體能夠分別作為人類先天性鐵粒幼細(xì)胞性貧血癥(CSA)、肝性紅細(xì)胞生成性卟啉癥(HEP)、雜色卟啉癥(VP)、細(xì)胞生成性原卟啉癥(EPP)的疾病模型,但其他幾種卟啉癥還沒有可穩(wěn)定遺傳的斑馬魚疾病模型。本文運(yùn)用TALEN技術(shù)建立了斑馬魚alad基因缺陷的突變體,作為人類5-氨基酮戊酸脫水酶缺乏性卟啉癥(ALA dehydratase deficient porphyria,ADP)的疾病模型。還運(yùn)用CRISPR-Cas9技術(shù)建立了斑馬魚cpox基因缺陷的突變體,作為人類遺傳性糞卟啉癥(Hereditary coproporphyria,HCP)的疾病模型。對兩種突變體的表型分析顯示,與野生型相比,alad-/-和cpox-/-突變體的血紅素合成受阻,體內(nèi)血紅素含量顯著減少,血細(xì)胞發(fā)育異常,血紅蛋白含量顯著下降,中間產(chǎn)物異常累積,同時(shí)血紅素合成通路中的相關(guān)基因表達(dá)水平異常。此外,人ALAD m RNA和CPOX m RNA分別能夠解救斑馬魚alad-/-和cpox-/-的表型,說明alad和cpox的功能在人和斑馬魚中具有保守性。斑馬魚中存在兩個(gè)編碼血紅素合成第三步反應(yīng)酶的基因,分別為hmbsa和hmbsb。系統(tǒng)樹分析表明這兩個(gè)基因?yàn)閺?fù)制基因,是人HMBS基因的共同直系同源基因(co-orthologs)。本文運(yùn)用CRISPR-Cas9和TALEN技術(shù)建立了斑馬魚hmbsa和hmbsb基因缺陷的突變體,作為人類急性間歇性卟啉癥(Acute intermittent porphyria,AIP)的疾病模型,并對其表型進(jìn)行了分析。兩種純合突變體均顯示血紅素缺失的貧血癥狀,并且突變體表型的顯著程度隨等位基因的缺失程度而增加。同時(shí)與野生型相比,兩種突變體幼魚和成魚的行為均顯示異常。為了對卟啉癥的病理機(jī)制做出更詳細(xì)的解析,本文還對兩種已有報(bào)道的斑馬魚卟啉癥及貧血模型urod-/-和alas2-/-進(jìn)行了高通量深度測序轉(zhuǎn)錄組分析,結(jié)果顯示血紅素的缺失導(dǎo)致大量基因的表達(dá)發(fā)生了改變。不同發(fā)育階段和不同突變體中的差異表達(dá)基因也不盡相同,顯示了卟啉癥的復(fù)雜性。血紅素合成的限速酶基因alas1的表達(dá)受生物鐘系統(tǒng)調(diào)控,生物鐘系統(tǒng)通過血紅素偶聯(lián)能量代謝途徑。為了深入探究生物鐘系統(tǒng)與血紅素合成的相互作用關(guān)系,本文還運(yùn)用CRISPR-Cas9技術(shù)建立了斑馬魚per1a基因的突變體以及per基因全部敲除的突變體。并證實(shí)了alas1的表達(dá)節(jié)律在per1a突變體中發(fā)生了改變。本文首次運(yùn)用反向遺傳學(xué)技術(shù)建立了四種人類卟啉癥的斑馬魚疾病模型,分別模擬人ADP,HCP和AIP,為相應(yīng)疾病的病理機(jī)制研究提供了重要的動(dòng)物模型,并為治療卟啉癥的藥物篩選工作提供了有力的工具。本文還建立了生物鐘突變體,為研究斑馬魚生物鐘系統(tǒng)與血紅素代謝間的相互作用關(guān)系提供了獨(dú)特的方向。
[Abstract]:Heme (heme) is an important molecule in the organism, which is involved in the formation of hemoglobin, myoglobin, cytochrome P450, vitamin B1 and so on. The synthesis of heme is composed of 8 steps enzyme catalyzed cascade reaction. The corresponding catalytic enzymes are encoded by corresponding genes, including ALAS1 and ALAS2, ALAD, HMBS, UROS, UROD, CPOX, PPOX and FECH. The process of synthesis of defects in heme enzyme function leads to a series called porphyria (porphyria) syndrome. The clinical symptoms of porphyria complex, diagnosis and treatment method at present is relatively limited. Zebrafish as a model organism, is widely used in the study of human disease, the corresponding gene deficient mutants can be used as a model of human disease porphyria. There have been reports of zebrafish ALAS2, UROD, PPOX, fech deficient mutants can respectively as human congenital sideroblastic anemia (CSA), hepatoerythropoietic porphyria variegate porphyria (HEP), (VP), cell generative protoporphyrin (EPP) disease model, zebrafish model but several other diseases there is no stable genetic porphyria. This paper uses TALEN technology to establish the zebrafish ALAD gene deficient mutant, as human 5- aminolevulinic acid dehydratase deficiency porphyria (ALA dehydratase deficient porphyria, ADP) disease model. Using CRISPR-Cas9 technology to establish the zebrafish CPOX gene deficient mutant, as human hereditary coproporphyria (Hereditary coproporphyria HCP) disease model. Two kinds of mutant phenotype analysis showed that compared with wild-type alad-/- and cpox-/- mutant heme synthesis is blocked, the body heme content decreased significantly, the abnormal development of blood cells, hemoglobin content decreased significantly, the intermediate product at the same time as abnormal accumulation of heme related gene pathway in abnormal expression. In addition, human ALAD m RNA and CPOX m RNA can save the phenotype of zebrafish alad-/- and cpox-/-, indicating that the functions of ALAD and CPOX are conserved in human and zebrafish. In zebrafish, there are two genes that encode heme to synthesize third - step reactivity enzymes, hmbsa and hmbsb, respectively. Phylogenetic analysis shows that these two genes are replicating genes and are common direct homologous genes (co-orthologs) of the human HMBS gene. This paper uses CRISPR-Cas9 and TALEN technology to establish the zebrafish hmbsa and hmbsb gene deficient mutant, as human acute intermittent porphyria (Acute intermittent, porphyria, AIP) of the disease model, and analyzed its phenotype. The two homozygous mutants all showed heme deficiency anemia, and the phenotype of the mutant was significantly increased with the degree of loss of allele. At the same time compared with the wild type, two mutants of juvenile and adult fish showed abnormal behavior. In order to make the analysis more detailed pathological mechanism of porphyria, the two kinds of reported porphyria and anemia model urod-/- and zebrafish alas2-/- high-throughput deep sequencing transcriptome analysis, results show that heme deficiency results in a large number of gene expression changes. The difference in different developmental stages and different mutants of gene expression are not the same, showing the complexity of porphyria. The expression of the speed limiting enzyme gene ALAS1 in heme synthesis is regulated by the biological clock system, and the biological clock system is mediated by the heme coupling energy metabolism pathway. In order to further explore the interaction relationship between the biological clock system and heme synthesis, we also used CRISPR-Cas9 technology to establish the mutant of per1a gene in zebrafish and the knockout mutant of per gene. It was also confirmed that the expression rhythm of ALAS1 changed in the per1a mutant. In this paper, using reverse genetics technology established four models of zebrafish human disease porphyria were simulated for the first time, ADP, HCP and AIP, provides an important animal model for the study of pathological mechanism of the disease, provide a powerful tool for drug treatment and screening of porphyria. The biological clock mutants have been established in this paper, which provides a unique direction for the study of the interaction between the biological clock system of zebrafish and the heme metabolite.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R-332;R55

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Kauppinen R.,崔榮;卟啉癥[J];世界核心醫(yī)學(xué)期刊文摘(皮膚病學(xué)分冊);2005年06期

2 ;卟啉癥(文獻(xiàn)綜述提綱)[J];重慶醫(yī)藥;1976年05期

3 Bickers D R;徐參;;卟啉癥的治療及其作用機(jī)制[J];國外醫(yī)學(xué).皮膚病學(xué)分冊;1982年04期

4 李發(fā)美;LimCK;PetersTJ;;卟啉癥研究的進(jìn)展[J];國外醫(yī)學(xué).輸血及血液學(xué)分冊;1989年02期

5 孫秀燕;卟啉反相高效液相色譜法分析及在生物醫(yī)學(xué)上的應(yīng)用[J];沈陽藥學(xué)院學(xué)報(bào);1987年02期

6 茅昌保;;鹽酸四環(huán)素光感作用引起卟啉癥樣皮疹[J];國外醫(yī)學(xué)參考資料.皮膚病學(xué)分冊;1978年03期

7 Poblete-Gutiérrez P.;Wolff C.;Farias R.;Frank J. ;羅素菊;;1例智利男孩發(fā)生嚴(yán)重的光過敏和手指縮短:南美洲首例純合子雜色斑駁卟啉癥[J];世界核心醫(yī)學(xué)期刊文摘(皮膚病學(xué)分冊);2006年Z1期

8 周勤;田君;;急性間歇卟啉癥1例臨床護(hù)理[J];齊魯護(hù)理雜志;2012年04期

9 Armstrong D.K.B. ,Sharpe P.C. ,Chambers C.R. ,G.H. Elder,黨倩麗;肝紅細(xì)胞生成性卟啉癥:尿卟啉原脫羧酶基因錯(cuò)義突變與疾病的輕重及卟啉分泌類型[J];世界核心醫(yī)學(xué)期刊文摘(皮膚病學(xué)分冊);2005年03期

10 錢恒林;晚發(fā)性紅細(xì)胞生成性卟啉癥[J];國外醫(yī)學(xué).皮膚病學(xué)分冊;1990年05期

相關(guān)重要報(bào)紙文章 前2條

1 ;現(xiàn)代病理學(xué)揭秘“吸血鬼”[N];消費(fèi)日報(bào);2003年

2 越婕;飲酒飲出卟啉癥[N];健康報(bào);2006年

相關(guān)博士學(xué)位論文 前1條

1 張淑青;人類卟啉癥及貧血的斑馬魚疾病模型的建立與解析[D];蘇州大學(xué);2015年

，

本文編號：1345132