TSG101在1996年由Stanley Cohen實驗室所發現，以antisense的技術使NIH3T3細胞不表現tsg101，並且導致細胞轉形及轉形細胞會在裸鼠身上形成腫瘤，顯示TSG101具有抑制腫瘤的特性。但後來的研究發現，在人類的癌組織genomic DNA並無TSG101缺失的現象，顯然TSG101為非典型的腫瘤抑制基因。並有報告指出TSG101參與MDM2/p53迴饋控制，及細胞膜傳輸作用之調控，其詳細之調節機制及確切的功能則尚待釐清。基於上述研究發現，本研究擬先確定tsg101在小鼠各器官組織細胞中之表現狀況，並研究與功能調控相關的TSG101蛋白磷酸化狀態。首先我針對成熟小鼠各器官進行免疫組織化學染色及原位雜交之分析，各結果顯示，在許多種神經細胞、上皮細胞和分泌細胞可以偵測到tsg101的表現，tsg101在各組織皆有表現，但組織中各種類型細胞中，tsg101蛋白之表現的steady-state量的狀態不同。其次探討TSG101蛋白的磷酸化狀態之分析，實驗在in vivo方面，首先以＃820抗體進行免疫沈澱，分離純化細胞(ARO、COS-1)之內生性TSG1 01蛋白及轉染COS-1細胞後表現之外源性蛋白HA-TSG (1-390)、HA-TSG(1-291)，再經西方墨點法分析，以對磷酸化serine，threonine具有特異性之抗體，檢測TSG101蛋白是否含有這些磷酸化之胺基酸，結果顯示，細胞內生性及外源性TSG101蛋白皆帶有磷酸化serine 及threonine。In vitro方面，將GST-TSG(1-291)、GST-TSG(214-390)的融合蛋白，及含有His-tag TSG (1-391)、TSG(1-291)、TSG(214-39)、TSG(130-240)、TSG (233-316)等融合蛋白，與激酶cdc2、GSK3β、PKA和PKC進行反應，結果顯示cdc2、PKC可以磷酸化GST-TSG(1-291)融合蛋白，cdc2、GSK3β、PKC可以磷酸化GST-TSG (214-390)融合蛋白，證實TSG101有cdc2、GSK3β、PKC激酶之磷酸化位點，顯示TSG101蛋白所扮演的生物功能可能受到這些激酶之調控。

Functional inactivation of tumor susceptibility gene tsg101 leads to cellular transformation and tumorigenesis in mice. No genomic DNA deletion in TSG101gene in human cancer indicated TSG101 is not a typical tumor suppressive gene. TSG101 participates in the MDM2/p53 feedback control loop and the regulation of the cellular membrane trafficking. However, detail functional characteristics remains to be elucidate.
In this study, we explored the tsg101 expression in adult mouse tissues from various organs using immunohistochemistry and in situ hybrid- ization. The results indicated that tsg101 expression was ubiquitous but in differential steady-state level in various cell types. The expression of tsg101 mainly found in epithelial cells、secretory cells and nerve cells. The second topic of this study was to characterize the phosphorylation status of TSG101 protein. Endogeneously expressed TSG101 and exogeneously expressed HA-tag TSG101 protein were purified by immunoprecipiation with #820 antiserum against TSG101, and were subjected for western blot analysis using anti-phosphoserine and anti-phosphothreonine antibodies. This experiment had confirmed that TSG101 protein contained both phosphoserine and phosphthreonine residues. In vitro kianse assay using GST-tag and his-tag TSG101 funsion proteins was exploited to investigate the kinase responsible for TSG101 phosphorylation. The results clearly indicated that cdc2、GSK3β and PKC kinases could phosphorylate TSG101 fusion Protein, implying that the function of TSG101 might be regulated by the signaling involving these kinases.

目錄
中文摘要………………………………………………1
英文摘要………………………………………………2
背景介紹………………………………………………6
材料與方法……………………………………………28
結果與討論……………………………………………38
參考資料………………………………………………42
圖表……………………………………………………61

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