臺灣博碩士論文加值系統

細胞爬行參與在各種重要的生理功能中，而細胞極性賦予細胞在爬行時的方向性。Focal adhesion是一包含integrin在內的蛋白質複合體，在細胞爬行時會由內而外連結細胞骨架以及細胞外基質。過去研究顯示integrin在胞囊運送至細胞膜的過程中是藉由Rab11來調控，但Rab11的調控機制至今未知。過去有研究指出最早在Saccharomyces cerevisiae中發現，且從酵母菌、果蠅、哺乳類及治人類中都具有高度保留性的蛋白質Mob2，可能透過Rab GTPase來調控胞囊運送。在此研究中，我們探討Mob2是否會透過Rab11調控integrin的表現，來影響纖維肉瘤細胞爬行。實驗結果顯示降低Mob2的表現會抑制纖維肉瘤細胞(HT1080)的爬行，Mob2會在細胞產生極性的過程中調節integrin的分佈。當Mob2過度表達在Rab11降解的細胞時，細胞的爬行則受到抑制，而當Rab11過度表達在Mob2降解的細胞時，細胞的爬行能力不會受到影響。本實驗接著發現miR758的過度表達則會誘發Mob2和Rab11的表現。綜合以上的結果，我們認為Mob2在Rab11所調控的integrin運送以及在細胞爬行上扮演重要的角色，而miR758則可能是在Mob2上游的位置調控Mob2的表現。他們之間的交互關係則須更進一步的實驗證明。

Cell migration is an important process which is involved in many biological functions. Focal adhesion, an integrin containing protein complex connected with extracellular matrix and cytoskeleton, plays the major role during cell migration. It has been reported that integrin is transported to cell membrane through vesicular trafficking and been regulated by Rab11, the endosome traffic protein. But how Rab11 been regulated is still unknown. Mob2, which has been first found in Saccharomyces cerevisiae, conserved from yeast, drosophila and mammals to human and is reported to regulate vesicular trafficking through Rab GTPase. Therefore, in this study, we investigated whether Mob2 could regulate sarcoma cell migration through Rab11 mediated integrin recycling. Results showed that Mob2 knockdown inhibited fibrosarcoma cell (HT1080) migration, and Mob2 regulated integrin recycling during the cell polarization. We further demonstrated Rab11 knockdown in Mob2 overexpressed cells inhibited cell migration; however, Rab11 overexpression in Mob2 knockdown cells has no effect on cell migration. This study also showed that miR758 overexpression induced Rab11 and Mob2 expression. Taken together, Mob2 may regulate Rab11-mediated integrin recycling for fibrosarcoma cell migration, and miR758 might play as a upstream regulator of Mob2, however, their causal relationship needs to be further investigated. 

致謝 1
Abstract 5
中文摘要 6
一、前言 7
1.1細胞爬行與細胞極性 7
1.2 Focal adhesion與integrin 8
1.3 胞囊運送與Rab11 GTPase 9
1.4 Mob2蛋白質(NDR，Cbk1，Rab) 9
1.5 miR758 11
1.6 研究目的 12
二、材料與方法 14
2.1 細胞培養 14
2.2 細菌轉殖 14
2.3 質體萃取 14
2.4 西方墨點法 15
2.5 細胞質體轉染 16
2.6 免疫螢光染色: 16
2.7 傷口癒合實驗 17
2.8 Transwell 細胞爬行實驗 17
2.9 Integrin recycle assay 18
2.10 免疫沉澱實驗 19
2.11 抑制Focal adhesion瓦解 19
2.12 數據分析 20
三、結果 21
3.1 Mob2表達量的下降降低人類肌肉瘤細胞HT1080的爬行能力 21
3.2 Mob2調控integrin的回送機制 21
3.3 Mob2透過Rab11調控細胞爬行 22
3.4 Mob2及Rab11在生化上的交互作用 25
3.5 miR758影響人類肌肉瘤細胞爬行 26
四、討論 28
4.1 Mob2影響人類肌肉瘤細胞HT1080的爬行能力 28
4.2 Mob2在integrin回送過程中的角色 29
4.3 Mob2與Rab11的上下游關係 30
4.4 miR758調控細胞爬行機制 31
五、參考文獻 35
六、圖表目錄 42
圖一、Mob2存在與否影響細胞移行 43
圖二、Mob2及integrin在分佈上具有重合性 44
圖三、Mob2的存在與否影響integrin的分布 45
圖四、Mob2不影響Rab11的表達量 46
圖五、Mob2及Rab11在細胞內的分佈 47
圖六、缺乏Mob2時Rab11極性運送受阻 48
圖七 Mob2的存在影響Focal adhesion與Rab11的分佈 49
圖八、Mob2透過Rab11影響細胞爬行 51
圖九、Mob2與Rab11具有生化上交互作用 53
圖十、miR758促進人類肌肉瘤細胞HT1080的爬行能力 54
圖十一、miR758促使Mob2及Rab11表達量上升 56
圖十二、Mob2在細胞爬行中的假設機制 57

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