Connexin29基因之研究 第一部份 Connexin29蛋白在成鼠耳蝸中的表現位置 第二部份 語言學習前期非症候群感音神經性聽障-Cx29基因突變之研究與功能分析

Study of the Connexin29 gene Part I Expression of gap junction protein connexin29 in the adult mouse cochlea Part II Mutation and functional study of Cx29 gene in prelingual nonsyndromic sensorineural deafness

廖珮如(Pei-Ju Liao)

指導教授 ： 李宣佑

中山醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

聽障(hearing loss)是一種常見的感音神經(sensorineural)異常疾病，聽障絕大多數都是因為內耳(inner ear)的缺陷而致病，在內耳中以耳蝸(cochlea)的構造對聽力的形成最為重要，connexins家族所構成的gap junction對於耳蝸內離子的?琠w性(ionic homeostasis)有很大的影響，故在聽障研究方面，connexin在耳蝸的表現及功能的探討為相當重要的一環。
在第一部份實驗中，我們想了解Cx29基因在老鼠耳蝸中的表現位置，故以免疫組織染色法(Immunohistochmistry)和雷射顯微擷取法(Laser Capture Microdissection)來調查Cx29的蛋白gje1(gap junction membrane channel protein epsilon 1)在老鼠耳蝸存在的組織部位，實驗結果發現gje1表現於stria vascularis、spiral limbus、spiral ligament、 organ of Corti部位，這些組織對耳蝸內淋巴液中高鉀低鈉的離子特性維持扮演著相當重要的角色。
第二部份我們想了解Cx29基因在遺傳性聽障中扮演的角色，故收集台灣地區250位語言學習前期聽障患者、26位自願性聽障患者，及120位聽力正常人血液DNA做Cx29基因的分析。結果發現了兩個多型性(polymorphism)：780+63 C→T heterozygous 和 840+2 T→G heterozygous，另外也發現三個突變點且均為heterozygous：一個錯意突變(misssense mutation) 807A→T/wt，會導致Cx29蛋白(GJE1)第269個胺基酸原為麩胺酸(Glutamic acid)被天門冬氨酸(Aspartic acid)取代，和兩個在intron的突變點780+15 G→A/wt 和780+10 G→C/wt。為了了解Cx29 807A→T錯意突變後產生的突變蛋白其功能上的影響，我們利用分子生物學的方法，建構正常及突變的Cx29蛋白表現質體轉殖進入HeLa細胞中，以了解Cx29突變基因的致病機轉，實驗結果證明正常的Cx29蛋白(GJE1)可以在細胞膜上形成gap junction，我們進一步以dye transfer實驗證明了GJE1所形成的gap junction確實具有細胞間物質流通的功能。但當GJE1的第269個胺基酸由麩胺酸以天門冬氨酸取代後，此突變蛋白堆積在細胞質內無法運送至細胞膜形成gap junction，此結果說明了Cx29 807A→T的錯意突變確實會對GJE1功能表現造成影響。
由上述的結果，我們推測Cx29在維持內淋巴液離子?琠w性上可能扮演重要的角色。本論文為首次發現Connexin基因族中E次群體的Cx29基因在老鼠耳蝸內表現位置，且可能和遺傳性聽障有關。

關鍵字

離子通道 ； 間隙連接 ； 學齡前遺傳性聽障

並列摘要

Hearing loss is one of the most common sensorineural diseases. The incidence of this profound prelingual deafness is approximately one per 1000 at birth. The cause of this disease includes many genetic and environmental factors, or both. Understanding of the genetic causes may provide the basis for neonatal and postnatal medical care to reduce the number of deaf baby. The sensorineural hearing loss is mostly resulted from damage to the inner ear, particularly cochlea. The gap junction formed by connexin is the critical element for maintaining the ionic homeostasis in cochlea. Therefore, it is important to study connexins in relative to the functional performance of cochlea and possible connexin alteration in hearing loss.
Previous study has shown that connexin gene Cx29 is required for gap junction formation in various tissue types in the mouse, but the expension of Cx29 and it’s precise functional performance in the cochlea remain unclear. We have initiated a study to investigate the expression of gje1(gap junction membrane channel protein epsilon 1), gene product of Cx29, in the mouse cochlea by immunohistochmistry and laser capture microdissection. We found that gje1 is present in the stria vascularis, spiral limbus, spiral ligament and in the organ of Corti, all of which presumably play important roles to maintain the ionic composition (high K+, low Na+).
We have determined the sequence in the coding region of Cx29 gene from 250 Taiwanese patients with prelingual deafness and 120 unrelated normal individuals. Two polymorphrisms, 780+63 C→T heterozygous and 840+2 T→G heterozygous, were observed in both deaf patients and normal individuals. Three mutations were found in the deaf patients, who are heterozygous for 807A→T, 780+15 G→A and 780+10 G→C, respectively. Protein encoded by Cx29 gene with 807A→T will result in a glutamic acid to aspartic acid substitution. To understand the role of 807A→T in pathogenesis, recombinant plasmids carrying either wild type Cx29 gene or 807A→T Cx29 have been constructed. These two plasmids were transferred into HeLa cells that are deficient in gap junctional communication. Expression of wtCx29 and Cx29 with 807A→T could be demonstrated, respectively, in the transfected HeLa cells. However, only HeLa cells traces duced with wtCx29 gained gap function and showed intercellular passage of lucifer yellow that allow exchange lucifer yellow between them. Our data indicate that the 807A→T in the Cx29 gene is defective in forming gap junction in HeLa cells.
To our knowledge this is the first demonstration of the association of a E connexin gene with human nonsyndromic deafness. Our study also implies that Cx29 may play an important role in the physiology of hearing, presumably by participating in the recycling of potassium to the cochlear endolymph.

並列關鍵字

connexin29 ； gap junction ； prelingual nonsyndromic sensorineural deafness

參考文獻

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