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研究生:李宜霖
研究生(外文):lee elin
論文名稱:Cbl-b的基因表現對全身紅斑性狼瘡及T細胞活化的影響
論文名稱(外文):The effects of cbl-b expression on SLE and T cells activation
指導教授:沈家瑞
指導教授(外文):Chia-Rui Shen
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:104
中文關鍵詞:T細胞活化cbl-bIL-2IL-2 promotersiRNA過渡表現
外文關鍵詞:T-cell activationcbl-bIL-2IL-2 promotersiRNAover-expression
相關次數:
  • 被引用被引用:1
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在T細胞活化中,cbl-b被發現可扮演一負向調控的角色。同時,在cbl-b基因缺失的老鼠中更發現,其T、B細胞會大量增生而導致嚴重的自體免疫疾病的發生。過去本實驗室發現全身性紅斑狼瘡(SLE)患者的cbl-b基因表現與其臨床症狀有高度相關性。因此,在本研究中,我們想探討cbl-b基因對T細胞活化(特別是IL-2)的影響。首先,我們建立可穩定以IL-2 promoter表現luciferase 的3T3/pIL2-Luc細胞株,並藉以大量表現cbl-b來了解cbl-b對IL-2 promoter的直接影響,結果發現當細胞株大量表現cbl-b,其luciferase的表現較與對照組比較下降34%。若改以Jurkat T細胞為細胞模式時,當同時轉殖入pIL-2/Luc與pCDNA3 cbl-b,細胞株之luciferase表現則可下降達80%。另一方面,當以可抑制cbl-b的表現之siRNA與pIL-2/Luc同時轉殖入Jurkat T細胞時,其luciferase的表現則相較於單純轉殖pIL-2/Luc與pCDNA3有顯著提昇的現象。而在IL-2的生成方面,也發現類似的結果,當過度表現cbl-b則IL-2產生量少,當以siRNA抑制cbl-b表現時,則可獲得較多IL-2的生成。因此,我們認為cbl-b的表現多寡可影響T細胞內IL-2 promoter的活性與IL-2的產生。而這樣的發現,或許在未來可應用在探討T細胞過度活化的SLE病人,了解cbl-b在其T細胞活化中所影響的訊號傳遞路徑。
Recent studies have revealed a crucial role of the cbl-b protein as a negative regulator in T-cell activation. In cbl-b knockout mice, hyperproliferation of the mature T and B cells results in the development of spontaneous autoimmunity. In our previous study, the expression of cbl-b is highly associated with the disease severity of SLE. Therefore, here we intended to clarify the role of cbl-b in T-cell activation, particularly in IL-2 production. First, we have established the stable clone, 3T3/pIL2-Luc, which consistently produces luciferase by IL-2 promoter. Second, we have identified the effects of overexpression of cbl-b on IL-2 promoter by determining the luciferase activity. We found that overexpression of cbl-b by transfecting pCDNA3 cbl-b in 3T3/pIL2-Luc cells suppressed the luciferase activity. Up to 80% of suppression in the luciferase activity was found in Jurkat T cells transfected with both pIL2-Luc and pCDNA3 cbl-b if compared with that in Jurkat T cells transfected with pIL2-Luc and pCDNA3. Furthermore, transfection of two different cbl-b siRNA was shown capable of inhibiting the cbl-b expression as well as enhancing the luciferase activity. Additionally, overexpression of cbl-b also reduced IL-2 production in Jurkat T cells, and cbl-b siRNA increased the production of IL-2. In summary, in this study, we show that over-expression of cbl-b significantly suppressed the IL-2 promoter activity and IL-2 production, whereas the inhibition of cbl-b enhanced the IL-2 promoter activity and IL-2 production.
目 錄
指導教授推薦書……………………………………………...……… Ⅰ
口試委員會審定書…………………………………………...……… Ⅱ
授權書………………………………………………………..………….Ⅲ
中文摘要……………………………………………………..………….Ⅵ
英文摘要………………………………………………………...………Ⅷ
誌謝………………………………………………………...……………Ⅹ
第一章 研究背景………………………………………………………. 1
1-1全身紅斑性狼瘡(Systemic lupus erythematosus, SLE)….… 1
1-2紅斑性狼瘡的治療…………………………………..……….. 5
1-3 Casitas B-lineage Lymphoma(cbl)基因…………………….. 8
1-4 Cbl在T細胞活化中扮演的角色……………………………. 9
1-5單核苷酸多型性Single nucleotide polymorphism (SNP)......... 12
1-6 SNP的應用……………………...………………………..…... 15
第二章 研究動機與目的…………...………………………….…........ 18
第三章 實驗設計與流程……………………………………………… 19
實驗材料與方法………..…………………………..………… 21
第四章 實驗結果………..……………...………………….………... 35
Part I:Cbl-b可經由調控IL-2 promoter而影響IL-2的表現... 35
PartⅡ:建立可快速篩檢SLE病患單點多樣性的檢驗方法.….. 39
第五章 討論……………………..…………………..………………. 42
參考文獻……….…………………………………….………….…… 47
附圖…………………………………………………..………………. 58
附錄…………………………………………………..………………. 71
參考文獻
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