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Student Number 982204018
Author Ming-long Peng(彭明隆)
Author's Email Address No Public.
Statistics This thesis had been viewed 655 times. Download 10 times.
Department Life Science
Year 2011
Semester 1
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Functional antagonism of proton-sensing G-protein-coupled-receptors
Date of Defense 2011-10-12
Page Count 101
Keyword
  • G-protein-coupled-receptors
  • proton
  • psychosine
  • Abstract T-cell death-associated gene 8 (TDAG8) is a member of proton-sensing G-protein coupled receptors, which are sensitive to acid stimulation. TDAG8 can also respond to the lysophospholipid, 1-β-D-Galactosylacyosylsphingosine (Psychosine), an intermediate of cerebrosides biosynthesis. A model was proposed in which the receptors have two ligand-binding sites, one for protons and the other for lipids. The liplids were suggested to interact with both sites, as agonist and antagonist, respectively. The aim of this study is toinvestigate whether psychosine acts on TDAG8 as an agonist and antagonized proton response. We stimulated cells with psychosine in the presence or absence of proton. Cells were stimulated with psychosine in presence or the absence of proton. The results have demonstrated that psychosine in presence or the absence of proton. The results have demonstrated that psychosine can act on TDAG8. Psychosine can inhibit proton-induced [Ca2+] increase in N2A cells, and vice versa.
    Table of Content 第一章 緒論………………………………………………………………………………1
    1.1 G蛋白偶合受體 (G-protein-coupled receptors, GPCRs)…………………………2
    1.1.1生理功能及其重要性………………………………………………………………2
    1.1.2結構與功能性…………………………………………………………………2
    1.1.3活化機制與下游反應…………………………………………………………3
    1.1.4酸敏感G蛋白偶合受體(Proton sensing G-protein-coupled receptors)……4
    1.2受體的雙配對(Double-paring)機制…………………………………………………4
    1.2.1氫離子(Proton)………………………………………………………………5
    1.2.2溶血磷脂質(Lysophospholipid)………………………………………………5
    1.2.3 OGR1受體家族對氫離子的下游反應和功能性……………………………6
    1.2.4 OGR1受體家族對溶血磷脂質的下游反應和功能性………………………7
    1.3半乳糖腦苷脂和氫離子對於TDAG8之間的關係…………………………………8
    1.3.1半乳糖腦苷脂在生理上的功能及其重要性…………………………………8
    1.3.2 半乳糖腦苷脂下游的訊息傳遞……………………………………………9
    1.4 研究動機 ……………………………………………………………………………9
    第二章 實驗材料與方法……………………………………………………………………………11
    2.1實驗材料……………………………………………………………………………12
    2.1.1菌株…………………………………………………………………………12
    2.1.2細胞株………………………………………………………………………12
    2.1.3實驗藥品……………………………………………………………………12
    2.1.3.1購自 Sigma藥廠 ……………………………………………………………………12
    2.1.3.2購自 Invitrogen公司 …………………………………………………………………… 12
    2.1.3.3購自 Qiagen …………………………………………………………………… 12
    2.1.3.4購自 其他 …………………………………………………………………… 12
    2.2實驗方法……………………………………………………………………………13
    2.2.1表現質體的確認……………………………………………………………13
    2.2.1.1限制酵素分析 …………………………………………………………………… 13
    2.2.1.2瓊脂醣膠的製備及電泳 ……………………………………………………………………13
    2.2.2大腸桿菌的轉型作用(Transformation)……………………………………13
    2.2.3細菌的培養…………………………………………………………………14
    2.2.3.1細菌固體培養……………………………………………………………………14
    2.2.3.2液體培養 ……………………………………………………………………14
    2.2.3.3菌種保存 ……………………………………………………………………14
    2.2.4表現質體的製備……………………………………………………………14
    2.2.4.1小量的製備(mini-prep) ……………………………………………………………………14
    2.2.4.2大量的製備(midi-prep) ……………………………………………………………………15
    2.2.5細胞培養(cell culture)……………………………………………………………………16
    2.2.6轉染作用(Transfection)……………………………………………………………………16
    2.2.6.1玻片的前處理 ……………………………………………………………………16
    2.2.6.2轉染作用(transfection) ……………………………………………………………………17
    2.2.7嘌呤黴素(Puromycin)篩選細胞……………………………………………17
    2.2.8聚合酶連鎖反應(Polymerase chain reaction, PCR)………………………17
    2.2.8.1核醣核苷酸(RNA)的萃取 ……………………………………………………………………17
    2.2.8.2 DNAase I處理 ……………………………………………………………………18
    2.2.8.3 cDNA的合成 ……………………………………………………………………18
    2.2.8.4反轉錄-聚合酶素連鎖反應(RT-PCR) ……………………………………………………………19
    2.2.9細胞內鈣離子濃度的分析…………………………………………………19
    2.2.10鈣離子成像分析方法……………………………………………………20
    2.2.11以流氏細胞儀觀察sh-mTDAG8的沉默效率(knockdown efficiency)……………………………………………………………………20
    第三章 結果 ……………………………………………………………………21
    3.1 TDAG8對Psychosine的刺激有濃度的依賴性………………………………22
    3.2 N2A細胞會受到Psychosine的刺激而活化,並有濃度的依賴性……………22
    3-3 TDAG8會受到Psychosine刺激而活化………………………………………24
    3.4在EGTA環境下,N2A細胞在pH6.0刺激下會得到最大的反應………………24
    3.5在EGTA pH6.0的環境下,OGR1和GPR4會受到刺激而活化…………………25
    3.6 Proton引發的下游反應主要是Gq途徑,Psychosine則是Gi途徑……………26
    3.7在含有氫離子的環境下會抑制由Psyhocsine引起的細胞內鈣離子濃度的增加……………………………………………………………………26
    3.8氫離子刺激N2A細胞會抑制Psyhocsine引發的鈣離子濃度增加………………27
    3.9 Psyhocsine刺激N2A細胞會抑制氫離子引發的鈣離子濃度增加………………28
    3.10細胞重置五分鐘後,psychosine和proton仍然可以引發反應…………………28
    3.11 Proton和Psychosine會相互抑制,且Psychosine的抑制性有濃度的依賴性……………………………………………………………………29
    3.12透過流氏細胞儀無法觀察到shRNA-mTDAG8對mTDAG8基因沉默果……29
    3.13透過聚合酶連鎖反應shRNA-mTDAG8無法有效的抑制TDAG8 mRNA的表現…………………………………………………………………………………30
    3.14在N2A和HEK293T細胞上,利用shRNA-mTDAG8會抑制Psyhosine引發的反應………………………………………………………………………………31
    3.15 shRNA-mTDAG8-B1轉染到N2A細胞,pH6.0刺激反應上有受到影響…….32
    3.16以流氏細胞儀分析不含紅螢光蛋白shRNA-mTDAG8 的沉默(Knockdown)效率…………………………………………………………………………………32
    3.17以流氏細胞儀分析含有紅螢光蛋白shRNA-mTDAG8 的沉默(Knockdown)效率………………………………………………………………………………33
    3.18以RT-PCR分析mTDAG8基因的沉默(Knockdown)效率……………………34
    第四章 討論………………………………………………………………………………35
    4.1半乳糖腦苷脂為TDAG8的配體,並能有效刺激活化TDAG8受體,並走Gi的反應途徑…………………………………………………………………………36
    4.2氫離子引發小鼠神經瘤母細胞內的鈣離子的累積主要為OGR1受體,並走Gq反應途徑…………………………………………………………………………37
    4.3探討氫離子與半乳糖腦苷脂對N2A細胞相互抑制的情形 ……………………38
    4.4利用流氏細胞儀和RT-PCR沒有明顯觀察到shRNA-mTDAG8 對TDAG8基因沉默的結果,但功能性分析上卻有影響……………………………………………41
    4.5利用流氏細胞儀和RT-PCR沒有明顯觀察到shRNA-mTDAG8 對TDAG8基因沉默的結果,但功能性分析上卻有影響……………………………………………42
    第五章 參考文獻 …………………………………………… 45
    附錄 …………………………………………… 91
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    Date of Submission 2011-11-30

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