臺灣博碩士論文加值系統

細胞膜上分布著許多蛋白，其中有些可扮演受器或是動器的角色，幫助細胞間進行訊息傳遞，或開啟細胞融合機制。細胞膜上有許多富含膽固醇與鞘脂的區域，稱為脂質筏，此處聚集許多膜蛋白，而口型蛋白質 (stomatin) 是其中一個主要組成，且在身體的多數組織中都有口型蛋白質的存在。先前文獻中發現，大量表現口型蛋白質可誘導細胞產生融合，且細胞表現的口型蛋白質可以傳遞到別的細胞，促使不同細胞進行融合，產生多核細胞。口型蛋白質的結構可分為多個功能性區域，但那些區域參與細胞融合尚不清楚，因此，本篇論文欲探討哪些功能性區域與細胞融合有關。利用分子生物技術將口型蛋白質上的部分區域摘除，或進行點突變，再建構大量表現不同片段口型蛋白質的倉鼠卵巢細胞 CHO-K1 和人類鱗狀癌細胞 A431 之穩定細胞株，進而觀察特定區域被摘除是否影響其分布、外泌體釋出、以及促進細胞融合功能。實驗結果發現，當 C 端被摘除後，會影響口型蛋白的分布，增加外泌體的分泌，但卻降低多核細胞的產生。由於 C 端可能是口型蛋白質與其他蛋白質交互作用的區域，當摘除後，可能失去與其他蛋白質的交互作用能力，造成讓細胞融合的功能喪失，因此 C 端扮演著影響外泌體分泌及細胞融合的重要區域。

In cell membrane, lots of integral proteins play roles of receptors and effectors. Some of them are required to passage a series of signals in initiating cell fusion. Membrane proteins are abound in small regions named lipid rafts, where cholesterol and sphingolipid are enriched. Stomatin is one of the major proteins in lipid rafts and is widely distributed over the human tissues. Previous researches showed that stomatin overexpression may induce cell fusion, besides, stomatin could also be secreted to promote fusion of other cells and both result in increasing the multinucleated cell proportion. The protein structure of stomatin is composed of several functional domains, however, the link between each domain and its function is still not established. In this study, I construct several truncated stomatin with deletions in different functional domains or point mutations at the post translational palmitoylation or phosphorylation sites. These constructs are genetically fused with fluorescence proteins and expressed in CHO-K1 or A431 cells through the lentiviral system and their distribution, secretion, and efficiency to increase multinucleated cells are then analyzed. Results revels that the C terminal deletion significantly changes the distribution, increases secretion, and decreases multinucleated cell formation. Stomatin was reported to associate with other proteins through the C terminal regions, therefore, this functional loss could be due to the its inability to interact with other proteins. According to these results, the C terminal region is crucial in its secretion and promoting cell fusion.

致謝......i
目錄......ii
Abstract......iv
摘要......v
圖表清單......vi
表格清單......vii
1. 緒論......1
1.1 口型蛋白質(Stomatin)......1
1.2 口型蛋白質的分布與功能......1
1.3 口型蛋白質與細胞融合和外泌體分泌之相關研究......2
1.4 口型蛋白質結構上之功能性區域......3
1.5 口型蛋白質的功能性區域與細胞融合之研究......4
2. 材料與方法......5
2.1 細胞培養......5
2.2 質體建構......5
2.2.1 cDNA 相關資訊......5
2.2.2 引子設計與建構變異性口型蛋白質......5
2.3 慢病毒系統與感染......6
2.4 螢光影像與共軛焦顯微鏡......7
2.5 西方墨點法與分析......7
2.6 多核細胞形成與分析......8
2.7 外泌體分泌與收集......8
2.8 統計分析......9
3. 結果......10
3.1 由cDNA建構變異性口型蛋白質......10
3.2 不同區域剔除後的口型蛋白質分布情形......10
3.3 點突變後的口型蛋白質分布情形......11
3.4 口型蛋白質的不同功能性區域參與細胞融合作用......12
3.5 變異性口型蛋白質影響外泌體的分泌......12
4. 討論......14
4.1 不同區域剔除後的口型蛋白質分布情形......14
4.2 點突變後的口型蛋白質分布情形......15
4.3 口型蛋白質的不同功能性區域參與細胞融合作用......15
4.4 變異性口型蛋白質影響外泌體的分泌......16
5. 參考文獻......18
6. 附錄......36-39

圖表清單
圖一、由 cDNA 建構變異性口型蛋白質......20
圖二、不同區域剔除後的口型蛋白質分布情形......22
圖三、點突變後的口型蛋白質分布情形......25
圖四、不同變異性口型蛋白質在 CHO-K1 細胞表現情形......28
圖五、不同變異性的口型蛋白質形成多核細胞的能力之差異......30
圖六、外泌體分泌在不同變異性口型蛋白質中有差異......32

表格清單
表一、不同變異性口型蛋白質在細胞內的分布情形......34

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