透過您的圖書館登入
IP:3.239.149.56
  • 學位論文

富含白氨基酸小多醣蛋白的基質蛋白與透明蛋白在斑馬魚眼睛扮演角色之探討

The Role of small leucine-rich proteoglycan proteins (keratocan and lumican) in the Zebrafish Eye

指導教授 : 錢宗良

摘要


基質蛋白(keratocan)和透明蛋白(lumican)屬於富含白氨基酸小多醣蛋白家族(small leucine- rich proteoglycan (SLRP),是角 膜基質層中主要細胞外基質(extracellular matrix)的蛋白質 (keratin sulfate (KS) proteoglycan)。兩者在胚胎時期角膜之發育和長大之後維持角膜清析度是非常重要。斑馬魚為一理想之脊椎動物模式來研究從胚胎到長大階段基因之表現,實驗中以斑馬魚為動物模式來研究基質蛋白和透明蛋白兩基因並且定出它們的特性,並了解其功能。 首先研究基質蛋白基因(zkera)結構並定出它的特性,另外研究已找出基質蛋白基因中1.7Kb 長度是有效之起動子(promoter)借由基因重組技術和顯微技術注射至魚卵內使早期發育時期表現出綠色螢光(EGFP),並且發現一偽基質蛋白基因。使用設計之反義核酸(morpholino)來降低魚胚胎中之基質蛋白的蛋白質量,如同剔除特定基因老鼠的身上表現出不正常的表現型,來研究早期發育時期蛋白質的作用功能。研究早期發育時期之形態分析,更進一步探討它們的功能與作用機轉。結果顯示基質蛋白反義核酸注射魚卵內後有出現較高之死亡率、表現型則尚須仔細定出特性。並且發現此一現象是經由caspase-3 and caspase-8 路徑之caspase- dependent pathway 之細胞凋亡現象。斑馬魚保存了大部份基質蛋白基因外還發現新的功能。 第二部份是研究透明蛋白基因,它是脊椎動物中角膜基質層與鞏膜中主要細胞外基質(extracellular matrix)的蛋白質。透明蛋白基因被認為與人類的近視(axial myopia)疾病有關。選擇以斑馬魚為動物模式來研究透明蛋白在近視發展中所扮演之角色。首先定性分析透明蛋白基因,了解斑馬魚透明蛋白基因之結構並與人類與老鼠及其他生物物種透明蛋白基因比較其演化關係,並且為了了解透明蛋白基因啟動子promoter 之功能以綠螢光(EGFP)來研究其表現。為了了解在 早期發育時,探討角膜透明蛋白在早期發育時所扮演之角色和如何作用的機轉,以透明蛋白反義核酸注射使基因失去作用knockdown。透明蛋白反義核酸注射後其中眼睛方面最明顯、且有趣的表現型是眼球會增大突出,此一現象隨著斑馬魚的年紀增大而更明顯。眼球會增大突出之表現與近視模式頗為相像,並且利用電子顯微鏡來了解不同時期眼睛超微結構是否與透明蛋白基因剃除老鼠和高度近視有相關連性。更進一步實行藥物篩選(drug screen test)以治療近視之藥物(muscarinic receptor antagonists)治療因透明蛋白基因失去作用而引起之眼球增大突出使之不會眼球增大,以此嘗試建立以斑馬魚為近視之研究模式。

並列摘要


Keratocan and lumican, belong to the family of small leucine-rich proteoglycans (SLRPs), are the main extracellular matrix proteins of the corneal stroma. Both play a pivotal role in maintaining corneal transparency and function during development. The zebrafish (Danio rerio) is an ideal vertebrate animal model to be applied to characterize and molecular analysis the keratocan and lumican genes. At first, we isolated and characterized the zebrafish keratocan (zKera) gene. Human keratocan sequence was used to search zebrafish homologues. The zKera full-length genomic DNA and cDNA were generated via PCR of zebrafish genomic DNA and RT-PCR of total zebrafish eye RNA, respectively. The zKera spanning 3.5 kilobase pairs consists of two exons and one intron, and a TATA-less promoter. The zKera encodes 341 amino acid with 59% identity to its human counterpart and 57% to that of mouse keratocan. Like mouse and chick keratocan,zKera mRNA is selectively expressed in the adult cornea, however, during embryonic development, zKera mRNA is expressed in both the brain and the cornea. Interestingly, it is expressed mainly in corneal epithelium but few in the stroma. A pseudogene was proved by introducing a zKera promoter-driven enhanced green fluorescence protein (EGFP) reporter gene into fertilized zebrafish eggs. Using morpholino-antisense against zKera to knock-down zKera resulted in lethal phenotype due to massive caspase-dependent apoptosis, which was noted by a significant increase of active caspase-3 and caspase-8, in the developing forebrain area including eyes. This is different from mouse, for which keratocan deficient mice are viable. Taken together, our data indicate that mammalian keratocan is conserved in zebrafish in terms of gene structure, expression pattern, and promoter function. The lumican gene (Lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. We chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zLum) spans approximately 4.6 kilobases (kb) of the zebrafish genome. Like human (hLUM) and mouse (mLum), zebrafish Lum (zLum) consists of three exons, two introns, and a TATA box-less promoter at the 5’-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mLum, zLum mRNA is expressed in the eye and many other tissues, such as brain, muscle and liver as well. Transgenic zebrafish harboring an Enhanced Green Fluorescent Protein (EGFP) reporter gene construct downstream of a 1.7 kb zLum 5’-flanking region displayed EGFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zLum expression by antisense zLum morpholinos (MO) manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g., atropine, pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

參考文獻


Amemiya CT, Zhong TP, Silverman GA, Fishman MC & Zon LI (1999) Zebrafish YAC, BAC, and PAC genomic libraries. Methods Cell Biol 60, 235-258.
Antonsson P, Heinegard D & Oldberg A (1991) Posttranslational modifications of fibromodulin. J Biol Chem 266, 16859-16861.
Berghmans S, Butler P, Goldsmith P, Waldron G, Gardner I, Golder Z, Richards FM, Kimber G, Roach A, Alderton W & Fleming A (2008) Zebrafish based assays for the assessment of cardiac, visual and gut function--potential safety screens for early drug discovery. J Pharmacol Toxicol Methods 58, 59-68.
Braat AK, van de Water S, Korving J & Zivkovic D (2001) A zebrafish vasa morphant abolishes vasa protein but does not affect the establishment of the germline. Genesis 30, 183-185.
Britten RJ & Davidson EH (1971) Repetitive and non-repetitive DNA sequencesand a speculation on the origins of evolutionary novelty. Q Rev Biol 46, 111-138.

延伸閱讀