臺灣博碩士論文加值系統

TSG101最早由發現Stanly N. Cohen所發現，在該篇研究中顯示TSG101表現不論過多或過少皆會造成細胞癌化，而在後續的研究更深入的瞭解TSG101在分子層次的功能與影響。總總的研究皆顯示TSG101影響範圍廣泛，但在癌症的臨床檢體中，卻鮮少發現TSG101突變，有趣的是，乳癌、子宮頸癌、肝癌、肺癌等臨床檢體中均可發現多種TSG101的剪接變異體。本實驗室先前發現在乳癌檢體中，TSG101剪接變異體TSG101-Δ154-1054的出現與術後復發（systemic recurrence）相關；TSG101-Δ154-1054和TSG101-Δ133-447同時出現與術後復發則有更高的相關。
因此本篇研究建立穩定表現TSG101-Δ154-1054之細胞株，觀察這些細胞株的phenotype，發現一旦表現TSG101-Δ154-1054可增加soft-agar colony formation的能力，卻不改變生長速度與血清需求程度。而在分子層次上，則發現wildtype-TSG101些微增加。以western blot方式探討與細胞生長相關之蛋白可發現MDM2-p90與p21也些微增加，但此改變卻不影響p53與Rb；OP18亦不受影響；CDC2明顯減少，連帶p-CDC2也隨之減少。在soft-agar colony formation相關途徑中發現HER3些微減少，HER2則無變化，EGFR family的下游基因ERK1/2則不受影響，但survivin mRNA卻減少。若強迫細胞暫時表現TSG101-Δ133-447，更可發現survivin mRNA減少甚至消失。
總括本篇論文，我們建立了穩定表現TSG101-Δ154-1054的細胞株，發現此類細胞株之soft-agar colony formation能力上升，但生長速度與血清需求程度則不改變。而影響soft-agar colony formation之訊息傳導路徑，則有待更進一步的研究。

In 1996, Stanly N. Cohen found whether up- or down-regulation of TSG101 would cause tumorigenesis and several follow-up researches further elucidated the molecular mechanism of TSG101. Notwithstanding TSG101 are known as influential proteins, the mutation of TSG101 is barely found in clinical data. What is interesting is several TSG101 splicing variants are reported instead. Clinical data show TSG101 splicing variants are found in breast cancer, cervical cancer, hepatocarcinomas, and lung cancer. We previously found that in breast cancer the TSG101-Δ154-1054 correlates significantly with systemic recurrence (p=0.05). Moreover, the co-existence of TSG101-Δ154-1054 and TSG101-Δ133-447 shows even higher correlation with systemic recurrence (p=0.0028).
In this thesis, we built-up 3 stable clones which over-expressed TSG101-Δ154-1054, and then assessed the tumorigenic activity of these 3 clones. We found that in these clones, the ability of soft-agar colony formation was primarily enhanced while the growth-rate and serum requirement were slightly or even not changed. Based on the western blot, wildtype-TSG101s were stabilize by TSG101-Δ154-1054. And we investigated the relative protein in cell cycle regulation. MDM2-p90, and p21 were slightly increased, but p53, Rb and OP18 were not influenced. CDC2 were even decreased. In the sight of the increase in soft-agar colony formation, the specific splicing variant could also reduce HER3 but HER2. However, the downstream proteins ERK1/2 were not influenced. Additionally, transient expression of TSG101-Δ133-447 could even abolish survivin mRNA.
To sum up, TSG101-Δ154-1054 stimulated the soft-agar colony formation but didn’t accelerate the proliferation. The molecular mechanism of the soft-agar colony formation still needs more investigation.

口試委員會審定書.................................................................i
謝誌................................................................................ ii
摘要............................................................................... iii
Abstract........................................................................... iv
目錄................................................................................ v
圖次............................................................................... vii
附錄索引......................................................................... viii
緒論................................................................................1
一、乳癌介紹............................................................................. 2
二、TSG101 介紹........................................................................ 5
三、TSG101 促進癌化之方式........................................................... 8
四、TSG101 與乳癌之關係.............................................................. 8
五、研究動機與實驗目的................................................................ 9
研究材料及方法................................................................. 11
一、 研究材料.......................................................................... 12
第一節 實驗材料來源........................................................... 12
第二節 乳癌細胞株 (Breast cancer cell line) ................................ 14
第三節 乳癌檢體引子 (primers) ............................................... 14
二、 研究方法.......................................................................... 15
第一節 TSG101-△154-1054 表達載體的構築.............................. 15
第二節 TSG101-△133-447 表達載體的構築................................ 20
第三節 建立穩定表達TSG101-△154-1054 的細胞株..................... 21
第四節 細胞癌化程度分析 (Tumorigenicity assays) ........................ 25
結果.............................................................................. 27
一、 TSG101 剪接變異體表現載體之構築........................................... 28
第一節 TSG101-Δ154-1054 表現載體之構築............................... 28
第二節 TSG101-Δ133-447 表現載體之構築................................ 28
二、 表達TSG101-Δ154-1054 會促進乳癌細胞株的soft-agar colony formation的能力............................................................................. 29
第一節 篩選穩定表現TSG101-Δ154-1054 細胞株.......................... 29
第二節 穩定表現TSG101-Δ154-1054 細胞株之癌化程度分析............ 29
第三節 分析表達TSG101-Δ154-1054 之乳癌細胞中一些分子的status.. 30
討論.............................................................................. 31
圖... .............................................................................. 35
附錄.............................................................................. 47
參考文獻......................................................................... 55

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