臺灣博碩士論文加值系統

Notch接受子對於生物的發育過程扮演很重要的調控角色。Notch-1接受子的活化是透過Notch-1接受子細胞外區域與鄰近細胞的ligand接觸，造成Notch-1接受子經過蛋白水解反應而釋放出Notch-1接受子細胞內區域( N1IC, Notch1 intracellular domain)。Notch-1接受子細胞內區域移動至細胞核中與RBP-Jκ結合 ( RBP-Jκ是結合在DNA上的蛋白)，形成N1IC/RBP-Jκ複合體，並且取代RBP-Jκ/histone deacetylase複合體結合在DNA上，活化Notch訊息傳導的下游基因。之前本實驗室曾經利用yeast two-hybrid 方法尋找與Notch-1接受子結合的蛋白質，βII-tubulin在此實驗被證實與N1IC有結合關係。βII-tubulin在之前研究被發現存在C6 glioma cell、MCF-7 breast carcinoma cell、HeLa cervix carcinoma cell等細胞株的細胞核中(Keliang Xu, 2002)。在此，本論文欲證實βII-tubulin與N1IC的結合關係，並且探討βII-tubulin在Notch訊息傳導所扮演的角色。首先，本論文在K562以及HeLa細胞株的細胞核偵測到βII-tubulin蛋白，並且進一步證實在HeLa細胞株的細胞核中，細胞核內的βII-tubulin會與Notch-1接受子有結合關係。以taxol處理K562以及HeLa細胞株後，經由CBF-1媒介的Notch-1訊息傳導會受到活化；以colchicine處理細胞則不影響經由CBF-1媒介的Notch-1訊息傳導。此外，以taxol處理K562以及HeLa細胞株，會造成細胞核內βII-tubulin、α-tubulin蛋白量的上升，以colchicine處理細胞則不影響細胞核中的βII-tubulin、α-tubulin蛋白含量。經由不同時間點的實驗證實經由taxol處理而進入細胞核的βII-tubulin、α-tubulin可以穩定存在細胞核中。以上結果顯示細胞核內βII-tubulin與Notch-1接受子在癌細胞細胞核內有結合關係，並且可以調控Notch-1訊息傳導。

The Notch transmembrane receptors play important roles in cell fate decisions during development. Ligand binding initiates the signal through the proteolysis of Notch-1 receptor to generate the Notch-1 intracellular domain (N1IC). The N1IC translocates into the nucleus to target the DNA-binding protein RBP-Jκ. The N1IC/RBP-Jκ complex overcomes the transcriptional repression by replacement of the RBP-Jκ/histone deacetylase complex to activate the target genes. Previously, we used yeast two-hybrid assay to identify proteins which associate with Notch-1 receptor, and βII-tubulin was identified to associate with N1IC. βII-tubulin was detected in the nuclei of C6 glioma cell, MCF-7 breast carcinoma cells, HeLa cervix carcinoma cells, etc (Keliang Xu, 2002). The aim of this paper was to characterize the roles of the nuclear bII-tubulin in the modulation of Notch signaling. Herein, I found that βII-tubulin was localized in the nuclei of HeLa and K562 cells. Furthermore, βII-tubulin associated with N1IC in the nucleus of HeLa cells. The CBF1-mediated transactivation activity of N1IC was modulated by taxol in K562 and HeLa cells, but not by colchicine. The contents of both nuclear βII- and a-tubulins were elevated by taxol in K562 and HeLa cells but not by colchicine. In time course experiment, the induction of nuclear βII-tubulin by taxol was stable in the nucleus of HeLa cell. These results suggested that nuclear βII-tubulin associated with the activated Notch-1 receptor to modulate Notch signaling.

縮寫詞彙說明: I
第壹章、緒論 1
第貳章、材料與方法 9
第一節、細胞株與細胞培養 9
第二節、質體構築 9
第三節、TRYPAN BLUE EXCLUSION 法，計算細胞數 10
第四節、西方轉漬法 (WESTERN BLOTTING) 11
一、 細胞核及細胞質蛋白質的萃取 11
二、以Bradford method定量蛋白質濃度 11
三、西方墨點法 (硫酸十二酯鈉-聚丙烯醯胺凝膠Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) 12
第五節、免疫沈澱法 (IMMUNOPRECIPITATION) 13
一、protein A sepharose的製備 13
二、protein A sepharose與目標蛋白之抗體的結合 14
三、細胞蛋白的萃取(本實驗使用三種不同的溶解緩衝液進行免疫沈澱) 14
四、目標蛋白質的免疫沈澱 15
第七節、LUCIFERASE REPORTER ASSAY 15
第八節、TAXOL的配置與加藥方式 16
一、 以taxol處理K562細胞 16
二、 先以cycloheximide處理K562細胞，再以taxol處理K562細胞 16
三、 以taxol處理HeLa細胞 17
四、 先以cycloheximide處理HeLa細胞，再以taxol處理HeLa細胞 18
五、 taxol處理HeLa細胞不同時間 19
第九節、COLCHICINE處理細胞 20
一、 先以cycloheximide處理K562細胞，再以colchicine處理細胞 20
二、 先以cycloheximide處理HeLa細胞，再以colchicine處理細胞 21
第叁章、結果 22
一、利用共同免疫沉澱的方式證實內生性的N1IC和內生性的βII-tubulin之間的結合 22
二、利用西方墨點法證實HeLa以及K562細胞株的細胞核內存在βII-tubulin 23
三、利用共同免疫沉澱的方式證實在細胞核中，內生性的N1IC和內生性的βII-tubulin之間的結合 23
利用西方墨點法證實HeLa以及K562細胞株的細胞核內存在βII-tubulin，因此接下來欲證明在HeLa細胞的細胞核中，內生性的N1IC和內生性的βII-tubulin具有結合關係。 23
四、利用癌症治療藥物taxol分析βII-tubulin是否影響經CBF-1媒介之Notch-1訊息傳遞 24
六、加入癌症治療藥物taxol，觀察細胞核βII-tubulin的變化 25
七、利用癌症治療藥物colchicine分析βII-tubulin是否影響經CBF-1媒介之Notch-1訊息傳遞 27
八、加入癌症治療藥物colchicine，觀察細胞核βII-tubulin的變化 28
九、以taxol處理HeLa細胞株後，在不同時間點收細胞，觀察細胞核βII-tubulin含量是否改變 28
第肆章、討論與分析 30
一、N1IC和βII-tubulin在細胞核內結合的情形 30
二、以抗癌藥物taxol處理K562以及HeLa細胞株，造成細胞核的βII-tubulin以及a-tubulin蛋白質含量上升，並且活化經由CBF-1媒介的訊息傳導路徑 30
三、以taxol處理HeLa細胞株，進入細胞核βII-tubulin蛋白，可以穩定的存在細胞核內 31
四、在不同種類細胞中，βII-tubulin存在於癌細胞細胞核的現象以及βII-tubulin進入細胞核的可能機制 31
文獻探討..................................................................................................34
附圖..........................................................................................................42

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