斑馬魚connexin基因之鑑定與功能分析

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 博碩士論文 > Item 310902500/4140

Please use this identifier to cite or link to this item: https://ir.csmu.edu.tw:8080/ir/handle/310902500/4140

Title:	斑馬魚connexin基因之鑑定與功能分析 Identification and function analysis of zebrafish connexin gene
Authors:	簡國軒 Kuo-Hsuan,Chien
Contributors:	中山醫學大學;醫學科技學院;生物醫學科學學系碩士班;李娟
Keywords:	班馬魚;內耳;通道蛋白 zebrafish;inner ear;gap junction
Date:	2011
Issue Date:	2011-10-25T07:02:31Z (UTC)
Abstract:	Cinnexin(CX)基因族所轉譯出來的蛋白質為家族蛋白質，它們會形成細胞間隙的連接通道(gap junction)。最近的研究指出CX基因的突變與一些人類疾病的發展有關，包括人類的聽覺障礙。在本研究中，我們探討了和人類與小鼠相似的斑馬魚cx基因之功能。我們以生物資訊的方法發現了和人類與小鼠GJC3(Cx30.2/Cx31.3)相似的斑馬魚基因cx43.4(zfcx43.4)。同樣的我們也找到和人類與小鼠GJB2(CX26)相似的斑馬魚基因cx30.3 (zfcx30.3)和cx27.5 (zfcx27.5)。藉由RT-PCR的分析我們可以發現這三個基因表現在斑馬魚成魚大部分的組織，包括內耳。同時觀察這三個基因在胚胎發育時期的表現，可以發現zfcx43.4基因在1.5 hpf到96 hpf有持續性的大量表現。而zfcx27.5則是微弱表現在1.5 hpf與3 hpf之時期，6 hpf之後到96 hpf則是表現量大量增加。相對的zfcx30.3則是在9 hpf才有偵測到微弱表現，直到25 hpf才有明顯的表現量。在全胚胎原位雜交(Whole-mount in situ hybridization)的結果可以發現zfcx43.4表現在72 hpf胚胎的中腦頂蓋(tectum)、後腦(hindbrain)、眼(retina)、鰓弓(Branchial arches)和胸鰭(pectoral fin)。zfcx27.5和zfcx30.3則是表現在胚胎耳囊(otic vesicle )。另外zfcx30.3在心臟也有表現訊號。同時我們製作zfcx30.3的多株抗體，並進行組織切片免疫螢光染色，我們發現zfcx30.3表現在胚胎72 hpf耳囊的聽斑(macula)下方的細胞核之間，還有心臟。在成魚內耳的球行囊(saccule)和卵形囊(utricle)聽斑，則可以看到zfcx30.3表現在支持細胞和毛細胞彼此的細胞核之間。我們使用zfcx30.3 反股Morpholino (MO)去抑制zfcx30.3在胚胎發育的功能，從morphant外型來看可以觀察到胚胎發生心臟水腫的機率增加。仔細觀察胚胎耳囊會發現耳石間距離變短，耳囊發育遲緩等現象。綜合以上所述，我們的研究成果將可提供一個好的基礎來進行後續的實驗。 The connexins (CXs) are a family of proteins that form gap junction channels. Recent investigations suggest that mutations of different CX have been identified in association with a wide variety of inherited diseases, including hearing loss. In this study, the functions of zebrafish cx genes are investigated. Our results indicated that cx43.4 gene of zebrafish (Zfcx43.4) is likely orthologous to human and mouse GJC3/Gjc3 using approaches of bioinformatics. Similarly, two zebrafish cx gene, cx27.5 (zfcx27.5) and cx30.3 (Zfcx30.3), are likely orthologous to human and mouse GJB2/Gjb2. All of three genes are expressed in most adult zebrafish tissues, including inner ear tissue using RT-PCR analysis. During embryogenesis, zfcx43.4 is consistent expressed from 1.5 hpf to 96 hpf by analysis of RT-PCR. Zfcx27.5 is small expressed in the 1.5 to 3 hpf and large expressed after 6hpf to 96hpf. Contrarily, zfcx33.8 transcripts are hardly detected until 9hpf and had a clear signal in 25hpf. Whole-mount in situ hybridization (WISH) revealed that zfcx43.4 is expressed in the tectum, retina, branchial arches, pectoral fin, and hindbrain of 72 dpf embryos. zfcx27.5 is major expressed in the otic vesicle. zfcx30.3 is detected in the zebrafish otic vesicle and heart. In addition, a zfcx30.3 polyclonal antibody is produced in our study. Our results indicated that zfcx30.3 is expressed in the region of under macula of otic vesicle and heart in 72hpf embryo using immunofluorescence stain. In addition, zfcx30.3 is detected in the hair cell and support cell of inner ear in adult zebrafish. Further, zfcx30.3 gene is knock down to observe the morphants phenytype using zfcx30.3 anti-sense Morpholino (MO). Our result indicated that morphants zebrafish increased the heart edema rate than wild type (WT) in 72 hpf embryo. In addition, our result found that otolith distance of morphants is shorter and delay of development than WT in 72 hpf embryo. These results will provide the basis for further study.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/4140
Appears in Collections:	[生物醫學科學學系暨碩士班] 博碩士論文

Files in This Item:

File	Description	Size	Format
index.html	碩士論文	0Kb	HTML	517	View/Open

Loading...