臺灣博碩士論文加值系統

中文摘要
前言：
心臟動作電位的發生和傳導與離子通道的活性、細胞與細胞間的傳導和心臟組織的結構有著複雜的關聯。近年來學者研究指出，Xin是一個Nkx2.5轉錄因子的下游基因，可能參與對心臟發育相當重要的BMP-Nkx2.5-MEF2C途徑。利用雙標記免疫螢光染色法 (double–label immunofluorescent )，發現Xin蛋白在胚胎時期即與β-catenin、N–cadherin有90﹪的重疊，在成鼠心臟介間盤的部分，Xin蛋白亦和connexin 43有80﹪重疊，顯示Xin蛋白表現於介間盤（intercalated disc）的adheren junction，在gap junction也可能有表現。因此心基因不僅對於心臟分化發育相當重要，對於心肌細胞間的連結、衝動傳導以及細胞骨架間的整合也扮演關鍵角色。因此心基因缺陷，可能造成介間盤結構缺損或是細胞間傳導障礙，相關研究亦顯示心基因缺陷小鼠會產生心肌肥大的現象。

實驗目的：
比較野生型小鼠（wild–type mice）與心基因缺陷小鼠 (Xin–deficient
mice；Xin+/- and Xin-/-) 之心室肌細胞動作電位、激發性心律不整和不正常
自動性節律發生機率，以及相關離子電流的表現，以利探討心基因陷缺小
鼠是否比較容易引發心律不整。
實驗方法：
一、 Xin基因型小鼠的鑑定
本實驗取小鼠尾巴做聚合酵素連鎖反應 (polymerase chain reaction, PCR)，以確定實驗用的每隻小鼠之基因型態。
二、小鼠心室肌細胞之分離
1. 將10–20週小鼠（體重為20–30公克）以sodium pentobarbital （50 mg/Kg i.p.）麻醉，經胸腔部開術取心臟出置於HEPES– Tyrode溶液中。
2. 以絲線將主動脈口固定於0.35 mm PE管的一端，另一端則與Langendorff灌流柱接合，以37℃ 100% 氧氣飽和灌流液進行冠狀動脈逆行性灌流，將心臟內之血液灌洗出，直至血液完全排出。
3. 以Ca2+–free HEPES– Tyrode溶液灌洗4–6分鐘，目的使心臟不再跳動、組織完全鬆弛。
4. 以Ca2+–free HEPES– Tyrode溶液含有collagenase （1 mg/ml）及protease（0.01 mg/ml）等兩酵素進行酵素溶液灌流，灌流約2–6分鐘不等。
5. 最後以Ca2+–free HEPES– Tyrode溶液灌洗10分鐘，減少酵素作用。分離出單一細胞後，逐次以HEPES–Tyrode溶液將Ca2+–free HEPES–Tyrode溶液更換掉，將鈣離子濃度回昇至生理常態濃度。

三、單一心室肌細胞之電生理研究
本實驗採whole–cell patch–clamp 方法，以電位箝定觀察三種型態小
鼠之心室肌細胞各離子電流的變化；及電流箝定觀察動作電位之異
同。
四、實驗數據分析：利用unpair t-test、One-way ANOVA以及卡方檢定
（Chi-square）進行數據分析。

實驗結果:
一、 以實驗中記錄到的細胞電容，比較心基因缺陷小鼠與野生型小鼠心室肌細胞大小差異，結果野生型小鼠、心基因缺陷小鼠異質體及同質體細之胞電容依序為101.3±5.8 pF、104±6.6 pF、132.4±7.8 pF，同質體小鼠無論是和異質體或是野生型小鼠比較，結果顯示均達統計顯著差異。表示此時期心基因同質體缺陷小鼠之心室肌細胞有明顯肥大的情形。
二、 在電流箝定模式下1Hz的電刺激誘發動作電位。三組間比較，心基因缺陷小鼠再極化50﹪、90﹪的動作電位期間（APD50、APD90）與野生型小鼠比較皆有顯著延長。APD50：依序為9.4±0.5 ms、20.5±5.5 ms及16.7±2.9 ms；APD90：依序為96.2±10 ms、138.6±17.0 ms及127.7±9.9 ms。
三、 三組在電流箝定模式下，1 Hz的電刺激下皆可記錄到EAD（early after-depolarization）、DAD（delayed after-depolarization）以及自動性節律的情形。其中EAD發生的機率，心基因同質體缺陷小鼠（26/44）較野生型小鼠（7/24）高，且達統計顯著差異。
四、 電位箝定下，發現心基因同質體缺陷小鼠INa (sodium current)在-40 mV（-7.7±1.6 pA/pF比上-3.2±0.9 pA/pF）、-30 mV（-9.2±1.5 pA/pF比上-3.9±2.5 pA/pF）電流密度較野生型小鼠大。Iti（transient inward current）之電流密度亦較野生型小鼠大（0.6±0.1 pA/pF比上0.4±0.1 pA/pF）；順向INCX電流密度較小，且達統計顯著差異。而T型鈣離子流（ICa,T）則是三組皆無表現。

結論：
此心基因缺陷動物模式除了在心臟傳導可提供臨床相關研究，在心臟肥大、心律不整方面與其離子電流的改變，亦可提供臨床治療方針的參考。

ABSTRACT

Introduction
Xin, one of the downstream targets of the Nkx2.5 and MEF2C transcription factors, is a striated muscle-specific gene. Xin protein localizes to the adherens junctions and gap junctions of the intercalated discs. It may play important roles in cardiac morphogenesis, formation and maintenance of the intercalated disc and myofibril integrity. Xin may also participate in generation and propagation of cardiac action potential. Xin expression is significantly up-regulated in pressure-overload animals and deficiency of Xin may lead to spontaneous hypertrophy in the hearts.

Aim
The aims of the present study were to compare the action potential characteristics, triggered and automatic rhythms and the ionic remodeling of Xin-deficient ventricular myocytes with wild-type ventricular myocytes.

Material and Methods
a. Gene typing
PCR（polymerase chain reaction）was used to confirm genomic type of Xin-deficient mice.
b. Isolation of ventricular myocytes
Male mice (C57BL/6J, 20–30 gm) and age–matched male Xin-deficient mice (20–30 gm) were anesthetized with sodium pentobarbital （50mg/Kg i.p.） and the heart and lungs quickly removed and immersed in Tyrode solution. The heart was perfused in a retrograde manner via polyethylene tube connected to the aorta. The free end of the polyethylene tubing was connected to a Langendorff perfusion column for perfusion with normal Tyrode’s solution at 37 ℃. The perfusion was replaced with oxygenated Ca2+–free Tyrode’s solution, then was replaced with solution containing collagenase (1 mg/ml) and protease (0.01 mg/ml) finally. Afterwards, the ventricle was cut away from the atrium and lung and placed in a dissection chamber containing Ca2+–free oxygenated Tyrode’s solution. The piece of tissue was cut into fine pieces and shaken in 20 ml of oxygenated Ca2+–free Tyrode solution until single ventricular myocytes obtained.
c. Electrophysiological study
The isolated cells were placed in a chamber mounted on the stage of an inverted microscope and superfused with extracellular solution appropriate to each patch–clamp experiment. Only cells showing clear cross striations were used for experiment on following ion currents and membrane potentials by means of whole–cell patch–clamp technique.
We respectively used current-clamp and voltage-clamp mode to record action potential characteristics and ionic currents, including ICa,T, INa, INCX and Iti.
d. Statistical analysis：
One-way ANOVA, unpair t-test and Chi-square were used to analyze data.

Results
The presented study showed that：
（1） The membrane capacitance（Cm）of Xin-deficient ventricular myocytes was significantly larger than wild-type ones, which proved that Xin-deficient animal could exhibit cardiac hypertrophy.
（2） The current density of INa and Iti in Xin-deficient ventricular myocytes were significantly increased, and the current density of forward mode Ni2+-sensitive INCX was reduced as compared to that of
wild-type ventricular myocytes. ICa,T was absent among three groups.
（3） As to the action potential characteristics, we found out that action potential duration was significantly prolonged and the incidence of early after-depolarization（EAD） was significantly higher in Xin-deficient ventricular myocytes. However, the incidence of delayed after-depolarization（DAD）and automatic rhythms had no significant difference among three groups.

Conclusion
The Xin-deficient mice might be an ideal animal model for structure-related electrophysiological study on the genesis of cardiac arrhythmias in cardiac hypertrophy.

目錄
頁次
目錄------------------------------------------------------------------------------I
圖目錄--------------------------------------------------------------------------IV
表目錄--------------------------------------------------------------------------V
中文摘要-----------------------------------------------------------------------VI
英文摘要-----------------------------------------------------------------------X
第一章 緒言------------------------------------------------------------1
第一節 介間盤（intercalated disk）簡介-------------------------1
第二節 心基因 (Xin–gene) 簡介-----------------------------------2
第三節 不正常自發性節律以及激發性節律的介紹------------5
第四節 和動作電位相關的離子流簡介---------------------------7
第二章 研究目的-----------------------------------------------------11
第三章 材料與方法--------------------------------------------------12
第一節 實驗動物-----------------------------------------------------12
第二節 小鼠心室肌細胞之分離-----------------------------------12
第三節 玻璃微電極與實驗裝置-----------------------------------14
第四節 全細胞膜電位箝定-----------------------------------------14
第五節 電生理實驗方法--------------------------------------------16
第六節 實驗藥物及溶液--------------------------------------------17
第七節 鑑定小鼠的Xin基因型-----------------------------------19
第八節 實驗數據與統計--------------------------------------------23
第四章 實驗結果--------------------------------------------------------24
第一節 鑑定小鼠的Xin基因型-----------------------------------24
第二節 野生型小鼠與心基因缺陷小鼠之心室肌細胞動作電位
比較-----------------------------------------------------------24
第三節 野生型小鼠與心基因缺陷小鼠之心室肌細胞各離子電
流比較 ------------------------------------------------------27


第五章 討論------------------------------------------------------------38
第一節 心基因缺陷導致心肌肥大之探討---------------------39
第二節 心基因缺陷與野生型小鼠動作電位方面之探討
---------------------------------------------------------------40
第三節 心基因缺陷與野生型小鼠心室肌細胞鈉離子電流
之探討-----------------------------------------------------41
第四節 心基因缺陷與野生型小鼠心室肌細胞的鈉鈣交換
離子電流之探討------------------------------------------42
第五節 心基因缺陷與野生型小鼠心室肌細胞T型鈣離子
流之探討--------------------------------------------------44
第六節 心基因缺陷與野生型小鼠心室肌細胞的短暫內向
離子電流與激發性心律不整之探討------------------46
第七節 對心基因缺陷小鼠心室肌細胞離子電流特性研究之總結
---------------------------------------------------------------48
第六章 結論--------------------------------------------------------------49
第七章 參考文獻--------------------------------------------------------51

圖目錄
圖1 心臟 發育及其相關的基因流程示意圖--------------------------10
圖4–1 鑑定Xin基因型小鼠的聚合酵素連鎖反應 (PCR)圖-----30
圖4–2 電刺激下，小鼠心室肌細胞之典型動作電位圖-----------32
圖4–3 電刺激下，小鼠心室肌細胞記錄到的激發性心律不整-33
圖4–4 小鼠心室肌細胞鈉離子流之比較圖--------------------------34
圖4–5 小鼠心室肌細胞納鈣交換離子流之比較圖-----------------35
圖4–6 小鼠心室肌細胞T型與L型鈣離子電流之比較圖-------36
圖4–7 小鼠心室肌細胞短暫內向離子電流之比較圖--------------37


表目錄
表一 小鼠之心室肌細胞的動作電位參數比較----------------------31

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