臺灣博碩士論文加值系統

中文摘要
聽障可因遺傳基因突變或環境因素，或兩者兼之引起。約有1/1000嬰兒在出生時或在小孩早期(即語言學習前時期prelingual period)罹患重度聽障(severe or profound)，在已開發國家約有60%個案是遺傳因素所造成的。到目前為止已知有59個基因發生突變會造成遺傳性聽障，各基因的致病機制不盡相同，非常複雜。Connexin (Cx) 是個龐大的家族，它所構成的gap junction channels負責調控生理和發育過程及離子與小分子的交流。每六個Cxs 形成一個hemichannel(connexon)，再由兩個hemichannels組成一個gap junction。本研究主要是利用細胞模式來探討Cx29與Cx43基因突變造成聽障的致病機制。
首先我們利用分子生物學的方法，先選殖出正常的Cx29與Cx43的基因與建構Cx29(E269D)與Cx43基因突變(S69P、T326I與932 delC)的表現載體，並進一步的將這些表現載體送入HeLa細胞中。利用細胞免疫染色法觀察到正常和異常Cx29與Cx43蛋白的表現位置。另外，我們利用microinjection的方式將Lucifer yellow染劑注入兩個鄰近有gap junction形成的HeLa細胞中的其中一個，並在注入染料3~5分鐘後，利用螢光顯微鏡觀察染料的轉移情形，來探討突變是否造成gap junction功能的影響。
本研究的結果，在Cx29基因研究方面，我們利用含螢光蛋白為質體的基因重組技術，使用微脂粒法基因轉殖感染HeLa 細胞，再利用免疫螢光染色法觀察正常或突變Cx29在細胞內的表現位置，結果發現正常Cx29-EGFP 融合蛋白表現在細胞膜，而Cx29E269D-EGFP 融合蛋白堆積在細胞質，進一步的研究發現是堆積在內質網(ER)。使用tet-on雙邊蛋白表現系統實驗的結果顯示Cx29E269D 突變會對正常Cx29造成顯性抑制效應(dominant negative effect)，使正常Cx29蛋白與突變Cx29(E269D)蛋白共同堆積在細胞質。
在Cx43和ΨCx43 (psueudogene)基因研究方面，我們同樣利用HeLa細胞來研究正常或突變Cx43蛋白在細胞內表現的位置，本研究共分析Cx43基因的三個突變點：S69P(ΨCx43)，932delC(ΨCx43)和T326I(Cx43)。結果發現含有 S69P、T326I和932del這三個突變的表現質體所轉殖的HeLa細胞，其突變蛋白並沒有影響蛋白運送到細胞膜，即此三種突變蛋白和正常的Cx43蛋白一樣被運送到細胞膜上形成gap junction。進一步了解Cx43三種突變基因(S69P、T326I與932delC)對gap junction功能性的影響，我們利用Lucifer yellow染劑通透實驗，發現表現S69P與T326I的兩種細胞，其染劑無法通透相鄰細胞，顯示S69P與T326I兩種突變蛋白確實會影響gap junction的功能，在另一個表現932delC的細胞中染劑對部份相鄰細胞具有通透能力，然而還是影響到gap junction的功能。
綜合以上的結果，我們已經利用分子生物學和細胞生物學的方法，建構正常及突變的Cx29和Cx43質體並表現於HeLa細胞中及進行功能性的研究，本研究使我們更深入解Cx29與Cx43突變基因造成聽障的致病機轉。

Hearing loss, caused by gene mutations and environmental factors, is a common sensory disorder in the human population. In the developed countries, the incidence of congenital hearing loss is estimated at 1 in 1000 births, of which approximately 60% cases are attributed to genetic factors. To date, 59 auditory genes have been identified.
Connexin (Cx) belongs to a large gene family, and the products of Cx gene family constitute a gap junction channel responsible for regulation of the physiological and developmental process, involving the exchange of ions and small molecules. Every six Cxs compose a hemichannel (so called as connexon), and then two hemichannel to form an integral gap junction. This study applied cell model to determine the Cx29 and Cx43 gene mutations in the pathogenic mechanisms causing hearing loss.
To confirm the localization patterns seen in the immunolabeling assay, HeLa cells were transfected with Cx29 constructs that were directly ‘tagged’ with GFP or DsRed at the C-terminal end of the protein. The results reveal the Cx29WT-EGFP was expressed along apposed cell membranes in the fluorescent localization assay with a continuous staining. In contrast, the p.E269D- EGFP (missense mutation) resulted in the accumulation of the Cx29 mutant protein in the endoplasmic reticulum (ER) rather than in the plasma membrane. Co-expression of Cx29WT and Cx29E269D proteins by a bi-directional tet-on expression system demonstrated that the heteromeric connexon accumulated in the cytoplasm, thereby impairing the formation of the gap junction. Assuming these findings, we suggest that Cx29E269D has a dominant negative effect on the formation of the gap junction.
To determine the crucial relationship between wild type Cx43 and three of mutant Cx43 genes( S69P、T326Iand 932 del C) were cloned and their functions were analysed in HeLa cells. Localization assay of WT Cx43 reveals a typical punctuate fluorescence pattern of a gap junction channel between neighboring expression cells. Additionally, immunoblotting analysis of the transfectants confirms the production of mutant proteins, in which their distributions along appositional membranes are determined using immunofluorescent staining procedures. Furthermore, dye transfer assay results demonstrate that gap junctional intercellular communication (GJIC) is less in HeLa cells carrying mutant GJA1 orψGJA1 gene than in WT-expressing cell.
In summary, we use molecular biology methods to create the normal and mutant constructs containing Cx29 or Cx43 gene and determine the pathogenic effects of normal and mutant Cx29or Cx43 gene in transfected HeLa cells. Our data reveal the Cx29 and Cx43 gene mutations play causal roles in the pathogenesis of hearing loss.

謝誌 I
縮寫字對照表 III
中文摘要 1
英文摘要 3
序論 (Introduction) 5
材料與方法 (Materials and Methods) 17
結果 (Results) 38
討論 (Discussions) 45
圖 (Figure) 53
表 (Table) 70
附圖(Suppl. Figure) 72
附表(Suppl. Table) 83
參考文獻 (References) 86
附錄 (Appendixes) 98
Paper

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