利用細胞模式探討GJB3基因突變造成非症候群聽障的機制

Functional study of GJB3 gene mutation in nonsyndromic deafness using cell model

柯姿羽(Tzu-Yu Ko)

指導教授 ： 楊建洲

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2012年)

https://doi.org/10.6834/CSMU.2012.00145

摘要

間隙連接 (gap junction) 是兩個細胞之間主司運輸的孔道，可容許分子量小於1千道爾頓 (dalton) 的物質通過。間隙連接基本組成單位為connexin (CX) 。Connexins (CXs) 基因家族在許多研究已經被報導和非症候群遺傳性聽障有關。在我們實驗室先前針對台灣地區513位非症候群聽障病患的篩檢中，已經發現在GJB3 (CX31) 基因中有六個錯意突變點，包括p.L10R、p.P18S、p.V84I、p.V174M、p.E183K及p.A194T。然而到目前為止對於這些錯意突變，所造成的影響及致病機轉並不清楚。因此在本論文中我們將分四部分加以探討。第一部分我們透過免疫螢光染色法分析正常及突變CX31蛋白在子宮頸癌細胞 (HeLa cells) 的表現位置。我們發現正常的CX31蛋白可以正常的表現和被運送到細胞膜，並在兩個鄰近細胞間形成間隙連接 (gap junction) 。另外，我們發現含p.V84I或p.A194T的CX31突變蛋白，其表現位置和正常的CX31蛋白一樣可被運送到細胞膜上。相反的，含p.P18S、p.V174M或p.E183K的CX31突變蛋白卻會堆積在細胞質中，無法正常送至細胞膜上表現。第二部分因為實驗室所發現帶有CX31錯意突變的病患多半屬於異質結合的基因型，所以我們利用共轉殖實驗 (co-transfertion) 探討這些突變蛋白是否會影響正常CX31蛋白在HeLa細胞內的表現位置。在共轉殖實驗中我們發現含p.V84I或p.A194T的CX31突變蛋白其表現位置會與正常的CX31蛋白疊合在一起，且共同表現在細胞膜上，這結果代表這些突變點並不會影響正常CX31蛋白的表現和運輸。為了進一步瞭解含p.L10R、p.V84I或p.A194T的CX31突變蛋白所形成的離子通道功能是否改變，我們利用刮痕附載染料轉移的技術分析間隙連接通道傳輸物質的能力。實驗結果首先我們證實實驗使用的HeLa細胞株本身無法通透染劑，這代表此株細胞不含內生性CX基因，這技術的建立將可幫助我們後續分析p. L10R, p. V84I或p.A194T的通透功能是否受到影響。在前面的研究中，我們發現轉殖入CX31V174M或CX31E183K突變的HeLa細胞會陸續的死亡，所以我們利用細胞存活檢測方法 (MTT assay) 分析細胞存活率。我們發現細胞如果含p.V174M或p.E183K的CX31突變蛋白，其還原MTT的能力有下降的趨勢，這代表細胞存活率會漸漸降低。綜合以上的結果，在本論文中我們已經瞭解p.L10R、p.P18S、p.V84I、p.V174M、p.E183K及p.A194T的CX31突變蛋白在HeLa細胞中的表現位置。另外，對於突變點p.V174M及p.E183K可能造成聽障的原因有一個初步的了解。這些結果讓我們初步的了解到CX31突變所造成的影響。未來我們需要更深入的針對這幾個突變點功能上的影響加以探討，以更清楚瞭解各突變點造成聽障的分子機制。

關鍵字

非症候群聽障 ； 錯意突變

並列摘要

Gap junctions (GJ) are groups of intercellular channels that allow transport of molecules with size less than 1KD. GJ channel is composed by connexin protein. Connexins (CXs) are known to be involved in human nonsyndromic genetic deafness. Recently, we have analysis the mutations of GJB3 (CX31) from 513 unrelated Taiwanese probands with non-syndromic hearing loss. We have identified six missense mutation, p.L10R, p.P18S, p.V84I, p.V174M, p.E183K and p.A194T, in the GJB3 gene of patients with hearing loss. However, the functional alteration of CX31 caused by the mutant GJB3 gene remains unknown. First of all, we want to know the expression and sub-cellular localization of wild-type CX31 or mutant CX31 protein using HeLa cells. CX31WT showed the typical punctuate pattern of gap junction channel between neighboring expression cells in the fluorescent localization assay. In addition, we have found that p.L10R, p.V84I and p.A194T mutant exhibited typical punctuate pattern of GJ channel between neighboring expression cells, which is similar to the CX31WT. Conversely, p.P18S, p.V174M or p.E183K mutants showed impaired trafficking of the protein to the plasma membrane and accumulation in the cytoplasm. Further, we simulate the heterozygous patients of CX31 missense mutations using co-transfection wild-type CX31 and mutant CX31 into HeLa cells. In co-expression study, p.V84I or p.A194T mutants was co-localized with wild-type CX31 and co-expressed in the membrane, respectively. Our results indicated that these mutants do not interfere CX31WT protein synthesis and transport to cell membrane. Moreover, we examine the functional change of gap junction intercellular communications (GJIC) in the HeLa cells with p.L10R, p.V84I or p.A194T mutants using scrape loading dye transfer. At least, we found that lucifer yellow (dye) was impermeated in HeLa cells. This confirmed that HeLa cells were gap junction-deficient cell line. The method created will help our further study. According to our previous result, we found that cells with p.V174M or p.E183K mutants were dead after transfected for 3 to 6 days. Therefore, we use MTT assay to determining viable cell number. We found that cells with p.V174M or p.E183K mutants were decrease ability to convert a soluble tetrazolium salt MTT into an insoluble formazan precipitate. Our results indicated that HeLa cells transfected with p.V174M or p.E183K may cause cell death. Based on these findings, we suggest that CX31P18S, CX31V174M and CX31E183K mutations have effect on the formation of GJ. Moreover, CX31L10R, CX31V84I, and CX31A194T mutations do not affect the trafficking of mutant CX31 proteins, but its functional significance remains unknown. Therefore, the functional significance of mutations requires further investigation.

並列關鍵字

CX31 ； GJB3 ； L10R ； P18S ； V84I ； V174M ； A194T ； E183K

參考文獻

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