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研究生:陳思帆
研究生(外文):Szu-Fan Chen
論文名稱:肌酸激酶調控鈉鈣交換蛋白機制之研究
論文名稱(外文):Study of the regulation of NCX1 activity by creatine kinase
指導教授:高閬仙高閬仙引用關係
指導教授(外文):Lung-Sen Kao
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生命科學暨基因體科學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:56
中文關鍵詞:肌酸激&肌酸激&肌酸激&肌酸激&
外文關鍵詞:NCX1creatine kinase
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鈉/鈣交換蛋白(Na+/Ca2+ exchangeer,簡稱為NCX)對於維持胞內鈣離恆定扮演著重要角色。到目前為止,哺乳類中已經發現了三種亞型的NCX,包括NCX1、NCX2以及NCX3。其中NCX1參與心肌細胞收縮,對於維持心肌功能很重要,然而當NCX1功能被抑制,卻可保護心肌細胞免於缺血/再灌流傷害(ischemia/reperfusion injury)。根據實驗室先前的研究,在HEK293T以及心肌細胞中,四種CK亞型中sMiCK和CKM會與NCX1交互作用。在細胞內低能量情況時,反向(reverse-mode) NCX1活性會降低,表現sMiCK和CKM可以恢復NCX1的活性。在本研究中,我建立了在HEK293T細胞中測量正向(forward-mode) NCX1活性的方法,並且發現了CKM與sMiCK參與調控正向NCX1活性。另外,在細胞低能量情況下,sMiCK可藉由保護粒線體免於斷裂達到調控粒線體生理功能,此一現象與粒線體內鈣離子調控無關。因此我的研究結果顯示CKM與sMiCK可調控NCX1正向與反向運輸的活性。
Na+/Ca2+ exchanger (NCX) is an essential component for maintaining Ca2+ homeostasis. Three mammalian NCX isoforms have been identified, including NCX1, NCX2, and NCX3. NCX1 plays an important role in cardiac muscle contraction; inhibition of NCX1 activity contributes to cardioprotection against ischemia/reperfusion injury. In our previous results two CK isoforms, sMiCK and CKM interact with NCX1 in HEK293T cells and cardiac myocytes. In addition, the decreased reverse-mode NCX1 activity under energy-compromised conditions is recovered by expression of sMiCK and CKM.In this study, I established the methods for measurement of the forward-mode NCX1 activity in HEK293T cells and demonstrated CKM and sMiCK also recovered the forward-mode NCX1 activity that decreased under energy-compromised conditions. Furthermore, sMiCK protected mitochondria from fragmentation but had no effects on the mitochondrial Ca2+ homeostasis under energy-compromised conditions. In conclusion, CKM and sMiCK can regulate both reverse- and forward-mode NCX1 activity under energy- compromised conditions.
Table of contents………………………………………………………………………1
中文摘要……………………………………………………………………………………3
Abstract……………………………………………………………………………………4
Abbreviation………………………………………………………………………………5
Introduction………………………………………………………………………………6
1. Na+/Ca2+ exchanger………………………………………………………6
1.1 Regulation of NCX activity…………………………………7
1.2 Pathophysiological roles of NCX…………………………8
2. Creatine kinase isozymes………………………………………………9
2.1 Functions of CK/PCr system…………………………………9
2.2 Pathophysiological roles of CK isozymes………………11
3. Mitochondria………………………………………………………………12
3.1 Mitochondrial dynamics and apoptosis…………………12
3.2 Mitochondrial Ca2+ transport……………………………14
4. Autophagy…………………………………………………………………14
5. Aims of this study………………………………………………………16
Materials and Methods…………………………………………………………………17
Metrials………………………………………………………………………17
Cell culture……………………………………………………………………………17
Transfection…………………………………………………………………18
Ca2+ image……………………………………………………………………18
Vector construction…………………………………………………………19
Reverse-mode NCX1 acitivity………………………………………………10
Forward mode NCX1 acitvity………………………………………………20
Mitochondrial morphology…………………………………………………21
Results …………………………………………………………………………………22
Locations of CKM and sMiCK in living cells…………………………22
Effects of CKM and sMiCK on forward-mode NCX1 activity…………23
Effects of sMiCK on mitochondrial morphology………………………24
Effects of sMiCK on mitochondrial Ca2+ homeostasis………………26
Autophagy in HEK293T cells………………………………………………26
Discussion………………………………………………………………………………28
Confromational change caused by GFP fusion…………………………28
Measurement of forward-mode NCX1 activity……………………………29
Relationship between mitochondrial dynamics and activities……30
Autophagy………………………………………………………………………31
References………………………………………………………………………………32
Figures……………………………………………………………………………………44
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