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研究生:李睿豪
研究生(外文):Jui-Hao Lee
論文名稱:口型蛋白質誘導多核細胞之形成研究
論文名稱(外文):Secreted Stomatin Can Trigger Multi-nucleated Cell Formation
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chi-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:55
中文關鍵詞:多核分泌
外文關鍵詞:stomatinmultinucleatedmitosisfusionsecrete
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口形蛋白質 (stomatin) 是屬於膜蛋白質且亦分佈於脂質筏 (lipid rafts) ,分子量大約是31.5 kDa,主要表現在身體的周邊血、肝臟、胎盤、肌肉、骨髓等部位。蛋白質之命名是來自人類的罕見遺傳性溶血性貧血 (rare overhydrated hereditary stomatocytosis) ,在病人的血液檢體發現其紅血球上的口形蛋白質之表現量與正常人相比較為減少甚至是短缺,但是從基因的分析發現其無突變的情況,過去的研究發現,口形蛋白質基因剔除小鼠亦沒有產生與人類相同的疾病症狀。因此,目前仍對於口形蛋白質之功能了解不多,雖然如此,對於口形蛋白質相似蛋白 (stomatin-like protein) 則有許多在過去的研究被提出其在生理功能上所扮演的角色。在線蟲模型上發現MEC-2與UNC-24分別在離子運輸與觸覺有關;在瘧原蟲上則是發現Pfstomatin與其感染紅血球有關;所以,我們也認為人類的口形蛋白質在其生理功能,應該也扮演著非常重要的角色。實驗室先前的發現是當細胞模型大量表現口形蛋白質時,會有較多的多核細胞形成,有些資料也暗示口形蛋白質可能會被細胞分泌並釋放到環境。在這篇報告,我們發現口形蛋白質為分泌性蛋白,利用免疫沉澱法收取培養基內的物質並證明其含有分泌性口形蛋白質,再利用插入式培養皿 (transwell) 進一步證明培養基內的確含有分泌性口形蛋白質,我們也發現此分泌性口形蛋白質可以直接和細胞膜或是細胞膜上之脂質筏的區域產生交互作用,進而進入細胞或是直接在細胞膜產生作用。最後,我們利用口形蛋白質轉染的倉鼠卵巢細胞 (Chinese Hamster Ovary - K1 Cells) 之培養基,即條件培養基 (condition medium) ,用來培養正常倉鼠卵巢細胞,同樣可以看到多核細胞的產生。此外,我們也初步發現這些多核細胞是如何形成,分析我們利用間歇攝頻技術 (time-lapse images) 所得到的細胞活動紀錄資料發現,這些多核細胞主要是透過細胞分裂時,細胞無法完成此程序並產生細胞融合 (furrow regression) ,另一部份是來自不同來源的細胞產生細胞融合 (cell fusion) 而產生。綜合以上發現,此篇報告發現口形蛋白質可被細胞分泌到環境,並且能夠直接與周圍鄰近之細胞的細胞膜區域產生交互作用,進一步使細胞形成多核細胞,報告中亦初步探討口形蛋白質造成之多核細胞主要是來自細胞融合。
Stomatin is a 31.5 kDa integral membrane protein or lipid rafts associated protein. It was named after the rare overhydrated hereditary stomatocytosis (OHSt) occurred in human. The distribution of human stomatin expression is major in peripheral blood, bone marrow, placenta, fetal lung, and fetal liver. Stomatin was absent or reduced from OHSt patients, but the mutated stomatin gene or stomatin gene knock out mice were not implicated any erythrocytes phenotype as OHSt patients. Although, the function of the stomatin is still unknown, there are some researches or proposals about stomatin orthologues or stomatin-like proteins. For examples, MEC-2, and UNC-24 in Caenorhablitis elegans; P-stomatin in Archaea; Pfstomatin in Plasmodium falciparum; some of the function of the orthologues have been proposed and partly elucidated, and the proteins are crucial for ion transport, mechanosensation, and parasites infection. Because the function of the human stomatin that we know little, even unknown, this short report will indicate the new role of stomatin in multi-nucleated cell formation, especially stomatin may present as secreted form. In the past study of our lab, cell models were transfected with stomatin gene, and we discovered the multi-nucleated cell formations occurred. In this report, I found the stomatin could be secreted to the extracellular medium by using immunoprecipitation and transwell assay to prove. In addition, the secreted stomatin could associate directly with the cell membrane or lipid rafts of the neighboring cells through transwell. Therefore, the condition medium containing secreted stomatin were used to treat CHO-K1 cells or HEK-293T cells, and the multi-nucleated cell formations were induced, too. Besides, our data suggested how the multi-nucleated cells formed. A series of time-lapse images were analyzed, and we indicated that the multi-nucleated cells were from abnormal cell mitosis, called post-mitotic cell fusion or furrow regression, and cell fusion. We conclude that the stomatin can be secreted to the extracellular medium and associate with the membranes of neighboring cells. The affected cells are induced to form multi-nucleated cells through furrow regression and cell fusion.
Acknowledgement i
摘 要 ii
Abstract iii
Index iv
Introduction 1
Materials and Methods 5
Cell Culture 5
Vector Construction, Transfection, and Infection 5
Reagents 5
Antibodies 6
Condition Medium (CM) 6
Immunoprecipitation (IP) and Western Blot Analysis 6
Immunofluorescent (IF) Staining of Cells Plated on Cover Slips 7
Coculture Cells for the Detection of the Secreted Protein 7
Multi-nucleated Cells Formation 7
CellTracker Assays 8
Time-lapse Confocal Microscopy 8
Cell Fusion Assay 8
Software for Quantification and Processing 8
Bioinformatic Website 8
Results 9
Stomatin Changes the Cell Morphology by Transfection 9
Stomatin Can Be Secreted to the Extracellular Medium 9
Secreted Stomatin-EGFP or Stomatin-RFP Can Associate with Cell Membrane 10
Stomatin Changes the Cell Morphology by Retroviral Infection 12
Secreted Stomatin Is Incorporated by the Cells 13
Secreted Stomatin Induces Multi-nucleated Cell Formation, Cell Fusion, Post-mitotic Cell Fusion, and Cell Internalization 13
Discussion 16
Figures 21
References 45
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