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系統識別號 U0026-1108201413320800
論文名稱(中文) CXCR4在調控T淋巴細胞移動方向性中所扮演的角色
論文名稱(英文) The role of CXCR4 in regulating the directional migration of T lymphocyte
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 102
學期 2
出版年 103
研究生(中文) 蔡怡瑩
研究生(英文) Yi-Ying Tsai
學號 s46014085
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-楊倍昌
口試委員-凌斌
口試委員-吳佳慶
口試委員-戴明泓
中文關鍵字 趨化因子受體CXCR4  T淋巴細胞爬行  化學趨化方向性  ZAP70 
英文關鍵字 chemokine receptor CXCR4  T lymphocyte migration  chemotactic directionality  ZAP70 
學科別分類
中文摘要 T淋巴細胞的遷移失常,而導致過度累積在發炎處,是造成慢性發炎的主要因素。T淋巴細胞遷移是由趨化因子(chemokine) 與其受體所調控的一個高度協調的過程。了解T淋巴細胞遷移的機制有助於釐清許多發炎相關疾病的病因。趨化因子受體CXCR4及其配體(ligand) – 基質細胞衍生因子(stromal cell-derived factor 1; SDF-1) 信號調控細胞遷移及附著力。ZAP70蛋白即是由CXCR4/SDF-1信號所活化的酪氨酸激酶(tyrosine kinase)。先前研究顯示,在SDF-1趨化物梯度下爬行的細胞,其ZAP70蛋白會不平均的分布在細胞前後兩端。一部分的ZAP70蛋白聚集在移動中的T細胞偽足前端,調控著T細胞趨化移動的方向性,另一部分則是聚集在尾端。在本篇研究中發現,SDF-1刺激會促成ZAP70和CXCR4形成複合體。並且在Jurkat與人類T淋巴細胞中觀察到hZAP70-EGFP融合蛋白的分布位置與CXCR4相近,且部分重疊。因此,我們認為在ZAP70蛋白聚集到細胞前緣促成偽足形成的過程,可能需要CXCR4。為了探討在細胞爬行的過程中,CXCR4與ZAP70位置的動態變化,我們建構hCXCR4與螢光蛋白DsRed結合的融合蛋白。在轉染至T細胞後,利用RT-PCR確認其表現。 hCXCR4-DsRed 的表現增加T細胞對SDF-1的穿透性的移動能力。表現hCXCR4-DsRed的細胞,在SDF-1刺激下,活化的ZAP70、ERK及1整合素增加。當hCXCR4-DsRed及hZAP70-EGFP融合蛋白同時轉染至T細胞,在爬行的細胞中,hCXCR4-DsRed的分布位置和hZAP70-EGFP重疊。透過cell^R 曠時影像系統觀察這些爬行的細胞可見,hCXCR4-DsRed首先聚集在細胞膜上,之後hZAP70-EGFP聚集到相近的位置,造成新偽足的形成。綜合以上結果,我們認為在SDF-1刺激下,CXCR4透過與ZAP70調控新偽足的形成,進而控制T細胞趨化移動的方向性。
英文摘要 Aberrant lymphocytes accumulation due to disregulation of T cell migration is a major cause of chronic inflammation. T lymphocyte migration is a highly coordinated process that is regulated by chemokines and its receptors. Identifying the mechanism of T lymphocyte migration may contribute to clarify the etiology of many inflammation- related disorders. Chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 (SDF-1) regulate cell migration and adhesion. ZAP70 protein is a tyrosine kinase activated by CXCR4/SDF-1 signaling. Our previous studies showed that ZAP70 protein accumulated unevenly in migrating cells in a chemoattractant SDF-1 gradient. A part of them is localized close to the protruding membrane of lamellipodia along with direction of migration, and the others are localized at the trailing edge. In this study, we found that ZAP70 bound to CXCR4 after SDF-1 stimulation. ZAP70 protein was co-localized with CXCR4 by CXCR4 antibody-induced patching of Jurkat and primary T cells. Therefore, we hypothesized that CXCR4 plays a role in the recruitment of ZAP70 protein at lemallipodia. To investigate the interaction between CXCR4 and ZAP70, we have constructed hCXCR4-hDsRed fusion protein to study the dynamic localization of ZAP70 and CXCR4 during T lymphocyte migration. The expression of pDsRed-hCXCR4 in both Jurkat and P116 T cells was confirmed by RT-PCR. Overexpression of CXCR4 increased the transmigration towards SDF-1 and induced ZAP70, ERK phosphorylation and 1-integrin activation, suggesting that the hCXCR4-DsRed protein is functional. We then co-expressed both hCXCR4-DsRed and hZAP70-EGFP fusion proteins in T cells and found that the distribution of hCXCR4-DsRed overlapped with hZAP70-EGFP during cell migration. By using cell^R time-lapse imaging system, we observed that hCXCR4-DsRed first located at the cell membrane then recruited hZAP70-EGFP and protruding lamellipodium was formed. In conclusion, CXCR4 plays a role in regulating the formation of lemellipodia and controlling the directionality of T cell migration through interaction with ZAP70.
論文目次 摘要 I
Abstract II
Acknowledgement IV
Contents VI
Figure index VIII
Introduction 1
Lymphocytes accumulation and inflammation disorders 1
T Lymphocytes migration to the site of inflammation 1
ZAP70 involves in T lymphocytes migration 3
Rationale of this study 4
Materials and Methods 5
Materials 5
Media and Buffer preparation 11
Methods 18
1. Transformation 18
2. Mini plasmid isolation 18
3. Cell culture 19
4. Isolation of primary T lymphocytes from peripheral blood 19
5. Reverse-Transcription Polymerase Chain Reaction (RT-PCR) 20
6. DNA construction 22
7. Transfection 23
8. Western‐blotting analysis 24
9. Cellular stimulation of ZAP70 and CXCR4 activity assay 26
10. Transmigration assay 26
11. Morphology and localization of CXCR4 and ZAP70 observation 27
12. Single cell migration assay 27
13. Immunoprecipitation 28
14. Statistical analysis 29
Results 30
CXCR4 recruited ZAP70 to the leading edge in migrating T cells 30
Construction and expression of human CXCR4‐DsRed fluorescence fusion protein in T cell lines 31
hCXCR4-DsRed enhanced the transmigration rate of T cells toward SDF‐1 in the presence of ZAP70 32
hCXCR4-DsRed enhanced the SDF-1/CXCR4 signaling in T cell lines 32
hCXCR4-DsRed co-localized with ZAP70 proteins in transfected T cells 33
hCXCR4-DsRed co-localized with the ZAP70 proteins at the leading edge of migrating T cells where new membrane protrusion occured 34
Discussion 35
Interaction between CXCR4 protein and ZAP70 protein in T lymphocytes 36
The role of CXCR4 protein in T lymphocytes migration 36
The function of CXCR4 protein in directional lamellipodia formation in T lymphocytes 37
References 39
Figures and Figure legends 44
Curriculum Vitae 57

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