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研究生:林育民
研究生(外文):Yu-Min Lin
論文名稱:探討活化型態的Notch1受體與其結合因子之結合生物功能
論文名稱(外文):Study on the biological function of the interaction between activated Notch1 receptor and its binding factors
指導教授:葉添順
指導教授(外文):Tien-Shun Yeh
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:細胞及分子生物研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:80
中文關鍵詞:Notch1受體
外文關鍵詞:Notch1
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中文摘要
Notch是個演化上高度保留,穿過細胞膜一次的受體蛋白,最早在果蠅中被發現。已知哺乳類的Notch受體蛋白分為Notch1∼4,位於細胞表面,被認為可調控基因的轉錄、細胞的增生、分化及早期細胞命運的決定,而Notch受體蛋白異常的活化會造成某些疾病的發生。當相鄰細胞表現的ligand結合在Notch受體蛋白的細胞外區域時,Notch訊號傳遞系統就被活化,使得整個細胞內區域在靠近內膜處斷裂,而形成活化型Notch (Notch IC, 細胞內區域)。此活化型Notch會轉移至細胞核中,並且和一些可與DNA結合的轉錄因子,如:RBP-Jκ/CBF1,以及其他細胞因子結合,進一步調控其標的基因的表現。由於這些參與的因子,對於Notch訊息傳遞路徑影響甚巨。因此,為了進一步探討Notch訊息傳遞路徑,本論文首要目標著重於探索參與Notch1下游訊息傳遞路徑仍未被發現的結合因子,及初步分析因結合所產生的生物功能。
在實驗的第一部分,我們利用K562、Jurkat、SUP-T1等人類細胞株證實了一個轉錄因子YY1與Notch1 IC (N1IC)之間的結合關係,同時也利用GST pull-down assay,找出N1IC和YY1 相互之間的結合區域,並藉由Sucrose gradient analysis證實細胞核內N1IC及YY1以大的complex結合形式存在,本論文也證實 YY1藉由N1IC與CBF1的直接作用結合在Wild type CBF1-response elements上,YY1會抑制CBF1媒介之Notch1訊息傳遞。同時,應用ChIP嘗試尋找未知受人類Notch1訊息傳遞所調控的下游基因。綜觀上述實驗結果,我們得知YY1藉由與N1IC結合,形成高分子量的複合體調控Notch 1訊息傳遞。
在實驗的第二部分,延續之前本實驗室已證實N1IC與b2-tubulin之間相互結合的結果,應用上述細胞分析N1IC與b2-tubulin之間的結合生物功能。實驗結果證實N1IC與b2-tubulin之間的結合是同時發生於細胞核及細胞質的,而應用RNA interference分析及細胞處理taxol的實驗結果更進一步證實b2-tubulin可能抑制經CBF1媒介之Notch1訊息傳遞。

Abstract
Notch receptors are evolutionally highly conserved and play important roles in modulating cell fate decisions throughout the development from invertebrate to vertebrate species. Four homologues of Notch receptors have been identified in human and aberrant Notch signaling is associated with a number of human diseases. There is a consensus model of Notch signaling pathway containing multiple proteolytic steps. After ligand binding, Notch receptors are cleaved to release the intracellular domains of Notch receptors. The intracellular domains, the activated form of Notch receptors, are then translocated into nuclei and interact with other transcriptional machineries to regulate the expression of cellular genes.
To dissect the molecular mechanisms of Notch signaling, the cellular targets interact with Notch 1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 by the pull-down assay of GST fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity trans-activated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human Jurkat cells and T-cell acute lymphoblastic leukemia cells. Additionally, we identified the cellular target genes modulated by the high-molecular-weight Notch complex by chromatin immunoprecipitation. Taken together, these results indicate transcription factor YY1 may modulate Notch signaling via association with the high-molecular-weight Notch complex.
We also demonstrated that the microtubule component 2-tubulin was associated with exogenous and endogenous N1IC in cells, Further study showed that N1IC and 2-tubulin associated in both nucleus and cytosol. The expression of 2-tubulin was inhibited by RNA interference and the nuclear 2-tubulin was depleted by the treatment of taxol to investigate the roles of 2-tubulin in Notch1 signaling pathway. The results suggested that 2-tubulin suppresses the luciferase reporter activity trans-activated by Notch1 signaling.

目錄
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.........................……..12
第四節、西方轉漬法(Western blotting)………………….....………….12
一、蛋白質的萃取與定量…………………………….…………12
二、硫酸十二酯鈉-聚丙烯醯胺凝膠電泳(Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)…...…...13
第五節、免疫沈澱法(Immunoprecipitation)…………….………..…...15
一、protein A sepharose的製備………………………...………15
二、 protein A sepharose與目標蛋白之抗體的結合…...………15
三、目標蛋白質的免疫沈澱................................………………16
第六節、融合蛋白的純化……………………………………………...16
第七節、GST fusion protein pull-down assay………………………….17
第八節、Protein-DNA binding assay…………………..………….……17
一、製備結合生物素之雙股DNA寡體…………........…..…..17
二、細胞核的萃取……………………….……………………...18
三、Protein-DNA binding assay……………………………..…..19
第九節、Sucrose gradient analysis…………………………..…………19
第十節、Luciferase reporter assay……………………………….…….20
第十一節、Chromatin Immunoprecipitation (1) ChIP (1)…………...20
第十二節、Chromatin Immunoprecipitation (2) ChIP (2)……………22
第十三節、RNA interference (RNAi)………………….………………23
第參章、初步結果……………………………….………………………24
第一節、應用本實驗室已建立之K562/pcDNA3、K562/HA-N1IC二種stable cell lines分析N1IC與YY1之間的結合關係…………..….24
一、利用共同免疫沈澱的方式證實外生性N1IC和內生性YY1之間的結合……………………………........................................24
二、 利用GST pull-down assay進行 mapping,找出N1IC和YY1相互之間的結合區域…..…………………..………………24
三、利用Sucrose gradient analysis分析N1IC和YY1在細胞核內結合的情形………………………………...………………….25
四、應用luciferase reporter assay分析YY1是否影響經CBF1媒介之Notch1訊息傳遞………………… …………………….26
第二節、應用Jurkat、SUP-T1兩種細胞及病人的T-ALL細胞分析N1IC與YY1之間的結合關係………………………………………27
一、確認Jurkat細胞表現內生性的N1IC和YY1…… ………27
二、利用Jurkat細胞及病人的T-ALL細胞以共同免疫沈澱的方式證實內生性N1IC和YY1之間的結合………………..……..27
第三節、利用Protein-DNA binding assay及ChIP (1),進一步去探討N1IC及YY1在DNA上可能的相互作用關係………….………….28
一、利用Protein-DNA binding assay證實YY1藉由N1IC與CBF1的直接作用結合在Wild type CBF1-response elements上……..28
二、應用ChIP (1) 確認由Protein-DNA binding assay所獲得的結果..............................................…29
第四節、應用ChIP (2)嘗試篩選未知受人類Notch1訊息傳遞所調控的下游基因…………………………….…..…….……………………29
一、藉由資料庫比對獲得DNA序列…………………………..29
二、利用luciferase reporter assay 分析N1IC對於具有受此篩選到的序列所調控的基因之影響…………………………………30
第五節、利用K562/HA-N1IC及SUP-T1二種細胞株分析N1IC與b2-tubulin之間的結合關係…………………………………………..30
一、利用共同免疫沈澱的方式證實於SUP-T1細胞內生性N1IC和b2-tubulin之間的結合………….………….…………………30
二、利用共同免疫沈澱的方式證實在K562/HA-N1IC細胞核及細胞質外生性N1IC與內生性b2-tubulin結合…………………………………………………………………31
三、利用Western blotting的方式分析內生性N1IC和b2-tubulin於SUP-T1細胞核內外的表現…………………….……………31
四、利用共同免疫沈澱的方式證實於SUP-T1細胞核及細胞質內生性N1IC和b2-tubulin之間的結合……….……………..…31
五、應用luciferase reporter assay及RNA interference分析b2-tubulin是否影響經CBF1媒介之Notch1訊息傳遞……….32
六、利用癌症臨床治療藥物taxol分析b2-tubulin是否影響經CBF1媒介之Notch1訊息傳遞…………………….……..…….32
第肆章、討論與分析…………………………………………………….34
一、在不同種類細胞中,N1IC與YY1之間的結合關係………..34
二、N1IC 藉由其ankyrin區域和YY1 結合……………………..34
三、分析YY1是否只靠其zinc finger區域就足以和N1IC結合..35
四、依據實驗結果,參考已發表論文,進一步分析N1IC和YY1在細胞核內結合的情形………………….…………………………35
五、YY1參與Notch1訊息傳遞所扮演的角色…………………...37
六、受Notch1訊息傳遞所調控的下游基因之篩選………………37
七、探討於RNA interference實驗中,轉染不同的RNA interference表現質體造成N1IC所活化的luciferase activity差異的可能原因.39
八、N1IC和b2-tubulin之間的結合………………………….…….39
參考文獻………………………………………………………………….42
表格……………………………………………………………………….47
附圖……………………………………………………………………….49

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