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系統識別號 U0026-0812200910353405
論文名稱(中文) FK506在離體青蛙心臟中抑制神經鈣磷酸酵素的去磷酸化作用而保護心臟功能的可能機轉
論文名稱(英文) The mechanism of cardioprotection of the FK506 inhibited calcineurin dephosphorylation in isolated frog heart.
校院名稱 成功大學
系所名稱(中) 生物學系碩博士班
系所名稱(英) Department of Biology
學年度 91
學期 2
出版年 92
研究生(中文) 黃秋堯
研究生(英文) Chiu-Yao (Paul)Huang
學號 l5685102
學位類別 碩士
語文別 英文
論文頁數 59頁
口試委員 指導教授-林進丁
口試委員-侯平君
口試委員-曾昌衍
中文關鍵字 虎皮蛙  鈉/氫離子交換子  心肌缺氧  總肌酸動酶活性  離體的青蛙心臟  缺氧後再灌流  神經鈣調磷酸酵素 
英文關鍵字 total creatine kinase activity  tacrolimus  Rana tigerina rugulosa  Na+/H+ exchanger  Na+ accumulation  myocardium  isolated perfusion heart  ischemic preconditioning  ischemia/reperfusion injury  ischemia  frog heart  FK506  cyclosporin A  cardioprotection  calcium overload  calcineurin dephosphorylation  calcineurin  working heart 
學科別分類
中文摘要 動脈硬化使心肌必須增加作功以維持正常的血液循環,導致心肌肥大或心肌缺氧,最後造成心衰竭。一般認為心肌缺氧導致鈣離子濃度增加,最後造成心臟功能受損。另一方面,當心肌增加做功時,心肌的鈣離子濃度上升,活化神經鈣磷酸酵素(Calcineurin),進而活化心肌增生基因。因此這兩種疾病都被認為與鈣離子有關,所以『是否神經鈣磷酸酵素也參與「缺氧後再灌流(Ischemia-reperfusion)」的心臟損害的過程』則為一個有趣而值得研究的題目。本篇研究是以離體的青蛙心臟為研究的材料,並以working heart的模式灌流。總肌酸動酶活性(Total CK activity)為心肌缺氧的指標;心律與形成的血壓乘積(RPP)為心臟功能的指標。由於因應不同的目的,心臟灌流分為三個部分。第一部分為紀錄當心臟經過缺氧(Ischemia)或缺氧預處理(Ischemic preconditioning, IPC)後不同時段總肌酸動酶活性的變化,它分為缺氧組及缺氧預處理組。第二部分為以高鈣離子灌流模擬「缺氧後再灌流」後過量鈣離子(Calcium overload)造成的傷害,它分為對照組、缺氧組、缺氧預處理組、高鈣離子灌流替代缺氧期組、高鈣離子灌流替代IPC加之20分鐘的缺氧期組及高鈣離子灌流替代IPC組。第三部分為施用神經鈣磷酸酵素抑制劑(CysA與FK506)對「缺氧後再灌流」的心臟損害的影響,有對照組、缺氧組、缺氧預處理組、CysA處理組與FK506處理組。
比較缺氧組與IPC組,在總肌酸動酶活性方面兩組之間並無顯著的差異,但在心臟功能指標(RPP)上證明的確IPC對心臟具有保護的作用。其次,以高鈣離子灌流模擬缺氧與IPC的實驗中,以過量鈣離子灌流20 min並沒有造成心臟功能的衰退;不過當以高鈣離子替代IPC灌流後,卻可以保護心臟不受缺氧期的影響。最後,以FK506抑制神經鈣磷酸酵素後,亦對心臟產生保護作用,並且直接降低總肌酸動酶的活性。根據本實驗的結果顯示:當心臟經過「缺氧後再灌流」後,對心臟造成損害的可能是在缺氧期時鈉/氫離子交換子(Na+/H+ exchanger)將鈉離子累積在心肌細胞中所造成的;另外,FK506抑制神經鈣磷酸酵素後所產生心臟保護作用,可能是透過抑制鈉/氫離子交換子功能的結果。
英文摘要 Myocardium ischemia induces calcium overload and resulting cardiac function damage. Calcium accumulation also can be found in cardiac hypertrophy, which is the result when Ca2+ activates calcineurin. Isolated frog hearts were used as subjects in this study and perfused with working heart method to determine if calcineurin is involved in ischemia-reperfusion induces damage. Total CK activity was used as an ischemic index and the product of heart rate and developed pressure (RPP) as a cardiac function index. The hearts underwent with different treatments in 3 sections, each with a different purpose. Section 1 observed for the time course of CK activity after ischemia and IPC in two groups — ischemia and IPC. Section 2 calcium overload was used to mimic ischemia-reperfusion injury in six groups — control, ischemia, IPC, Ca2+-substituted ischemia, Ca2+-substituted IPC with ischemia, and Ca2+-substituted IPC. Section 3 tested for the role of calcineurin inhibition by CysA and FK506 in cardioprotection in five groups — control, ischemia, IPC, CysA treatment, and FK506 treatment.
Comparison of total CK activity did not reveal any difference between ischemia and IPC groups, whereas the comparison of RPP did show cardioprotection in IPC treatment. High calcium concentration perfusion was used to mimic ischemia and IPC in section 2. Unexpectedly, the induced calcium overload over a period of 20 minutes did not result in heart damage. However, mimics IPC did induce cardioprotection. As the heart treated FK506 before ischemia could affect cardioprotection and decrease total CK activity directly. These results suggest that the main factor affecting cardiac function damage is accumulation of myocardial sodium by Na+/H+ exchanger during ischemia, whereas the inhibition of calcineurin of FK506 induces cardioprotection by inhibiting Na+/H+ exchanger.
論文目次 中文摘要 -------------------------------------- i
Abstract -------------------------------------- iii
Key Words ------------------------------------- v
Abbreviations --------------------------------- vi
Acknowledgements ------------------------------ vii
Contents -------------------------------------- ix
List of Tables -------------------------------- xi
List of Figures ------------------------------- xii
Introduction ---------------------------------- 1
Materials and Methods ------------------------- 5
Animals ------------------------------------- 5
Perfusion Procedure (Working Heart) --------- 5
Experimental Protocol ----------------------- 7
Section 1. Time Course of Total CK Activity
after Ischemia and IPC -------- 8
Section 2. Effects of Calcium Overload in
Ischemia and IPC -------------- 8
Section 3. CysA and FK506 Inhibit Calcineurin
Dephosphorylation in Ischemic
hearts ------------------------ 9
Total CK Activity Assay --------------------- 10
Statistics ---------------------------------- 11
Results --------------------------------------- 12
Omitting Outlying Value --------------------- 12
Generally Gender Differences ---------------- 12
Time Course of the Total CK Activity after
Ischemia and IPC ------------------------ 13
IPC Results Cardioprotective, but no Decreases
in Total CK Activity -------------------- 13
Different Patterns of Total CK Activity between
Male and Female hearts ------------------ 14
Calcium Overload in IPC Period Induces Cardio-
protective, but It does not Cause Cardiac
Damage in the Ischemic Phase ------------ 15
Calcineurin Dephosphorylation Inhibition by
FK506 Affecting Cardio- protection ------ 16
Discussion ------------------------------------ 17
Total CK Activity does not Decrease after IPC 17
Calcium Overload and Na+/H+ Exchanger (NHE) - 18
IPC Induces Cardioprotection and NHE Inhibition
Induces Cardioprotection ---------------- 20
The Role of Calcineurin in Cardioprotection - 22
References ------------------------------------ 25
Resume ---------------------------------------- 59
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