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研究生:莊雁婷
研究生(外文):Yen-Ting Chuang
論文名稱:幽門螺旋桿菌空泡細胞毒素中p37片段短暫地表現在中國蒼鼠卵巢細胞內引起細胞凋亡
論文名稱(外文):Transient expression of p37 fragment of Helicobacter pylori VacA protein induces apoptosis in Chinese hamster ovary cells
指導教授:劉銀樟
指導教授(外文):Yin-Chang Liu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
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:2004
畢業學年度:92
語文別:英文
論文頁數:56
中文關鍵詞:幽門螺旋桿菌空泡毒素細胞凋亡中國蒼鼠卵巢細胞綠色螢光蛋白質免疫沈澱
外文關鍵詞:Helicobacter pyloriVacuolating cytotoxinApoptosisChinese hamster ovary cellGreen fluorescent proteinImmunoprecipitation
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Vacuolating cytotoxin(簡稱VacA)是幽門螺旋桿菌(Helicobacter pylori, H. pylor)主要毒素之一,可能會引起慢性胃炎、消化性潰瘍及胃癌。本研究將VacA的主要毒性部分『p37』表現在中國倉鼠卵巢細胞(Chinese hamster ovary cell, CHO-K1)內,藉以尋找此毒素在哺乳動物細胞內的作用目標。首先,把從H. pylori 26695中得到的p37基因和哺乳動物表現質體(mammalian expression vector, pEGFP-N3)進行基因接合反應(ligation),讓細胞內製造出的p37與綠色螢光蛋白質(green fluorescent protein, GFP)形成融合蛋白質(p37-GFP fusion protein)。結果顯示,細胞內表現的p37-GFP主要聚集在細胞質,透過錐藍排除法(trypan blue exclusion assay)發現細胞存活率(cell viability)明顯地減少。分析細胞增生率(cell proliferation)和使用其他專門用來檢測細胞凋亡(apoptosis)的方法更加支持這項結果。另外,切除p37其N端32個氨基酸可有效地破壞p37的細胞毒性(cytotoxicity)。這項發現和先前研究認為VacA需要N端輔助才具有細胞毒性的結果一致。而且,在免疫沈澱(immunoprecipitation)實驗中發現一個大約70 kDa蛋白質會和p37產生交互作用(interaction),但GFP或 失去N端的p37-GFP(truncated p37-GFP, �愎37-GFP)並沒有和任何蛋白質有交互作用。根據以上實驗,我認為VacA的p37片段會和細胞內的蛋白質產生交互作用並引起細胞凋亡。
The vacuolating cytotoxin (VacA) is one of the major virulence factors of Helicobacter pylori (H. pylor), which may cause chronic gastritis, peptic ulcer and gastric cancer. In this study, p37, the principle catalytic domain of VacA was manipulated and expressed in Chinese hamster ovary cells (CHO-K1) to identify the mammalian cellular targets of the virulence factor. The p37 gene, derived from H. pylori strain 26695, was cloned into a mammalian expression vector pEGFP-N3 in a manner that p37 was expressed as a chimeric protein of green fluorescent protein (GFP). It was found that expression of p37-GFP, which localized mainly in the cytoplasm, was accompanied with a significant reduction of cell viability as determined by trypan blue exclusion assay. This observation was further supported by cell proliferation assay and the other analyses more specific to apoptosis. Deletion of the N-terminal 32 residues of p37 significantly abrogated the cytotoxicity of p37. This is consistent with the previous observation by other investigators that the N-terminal region is required for cytotoxicity of VacA. Furthermore, p37 but not the N-terminal truncate version, in the immunoprecipitation experiment, could interact with a cellular target with 70 kDa in molecular weight. In conclusion, p37 of VacA may interact with intracellular protein(s) and induce cell apoptosis.
Page
Abstract (in Chinese) I
Abstract (in English) II
Acknowledgment III
Abbreviations IV
Table of contents V

I INTRODUCTION 1
1.1 Helicobacter pylori 1
1.2 Vacuolating cytotoxin A 2
1.3 VacA-induced cell apoptosis 3
1.4 The aims of this study 4

II MATERIALS and METHODS 6
2.1 Cell culture 6
2.2 Cloning of p37-GFP and truncated p37-GFP 6
2.3 Transfection into CHO-K1 cells 7
2.4 Fluorescence microscopy 8
2.5 Flow cytometric analysis of fluorescence expression 8
2.6 Western blotting for detection of GFP fusion protein 9
2.7 Trypan blue exclusion assay 10
2.8 MTT assay for cell proliferation 10
2.9 Observation of nuclei morphology by staining with DAPI 11
2.10 Agarose-gel analysis for DNA fragmentation assay 11
2.11 Detection of apoptosis and cell cycle analysis 12
2.12 Immunoprecipitation 12

III RESULTS 14
3.1 Construction of p37-GFP and �愎37-GFP 14
3.2 Cytosolic expression of p37-GFP and �愎37-GFP fusion proteins 15
3.3 Cytosolic expression of p37 induced cell death through apoptosis 16
3.4 p37-GFP expressed in the cells localized to cytoplasm 19
3.5 Identification of p37-GFP interacting proteins by immunoprecipitation 20

IV DISCUSSION 21

V REFERENCES 26

VI FIGURES and LEGENDS 33

VII APPENDIX 47
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