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研究生：	劉閔禎 Ming-Chen Liu
論文名稱：	Effects of periodic and sustained stretching on cardiac culture
指導教授：	陳志強 Chi-Keung Chan
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 生物物理研究所 Graduate Institute of Biophysics
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	90
中文關鍵詞：	心臟、心肌細胞、拉伸、周期拉伸、持續拉伸、拉伸激活通道、機械力敏感通道、纖維母細胞
外文關鍵詞：	cardiac, stretch, periodic stretching, sustained stretching, stretch-activated ion channel, SACs, Gadolinium
相關次數：	點閱：14 下載：0
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一般來說我們認為心臟跳動是藉由電訊號傳遞來控制的。但是研究發現除了電訊號之外，機械力也能夠誘導心肌細胞上的機械力敏感離子通道開啟並且產生電流進而影響心臟的電活動。本實驗主要是探討機械力對心臟細胞跳動週期的影響，另外我們也探討纖維母細胞的數量多寡是否影響跳動週期。

心臟細胞是由大鼠胚胎（懷孕17天）的心臟取得，並且培養在可變形的腔室中，我們以單軸方向拉腔室。本實驗使用了兩種形式來拉伸細胞：(1) 週期性拉伸：1 Hz下持續二到四個小時，並以鈣離子螢光染色來觀察細胞跳動，(2) 持續性拉伸：將腔室維持在拉的情況下五分鐘再放掉腔室，並在明視野顯微鏡下觀測。本實驗使用了拉伸激活離子通道阻斷劑：釓(Gd3+)，這是一個應用在機械力敏感通道上常見的阻斷劑。

心臟細胞在週期性拉伸過後，我們發現細胞的跳動週期會接近拉伸的週期。
在持續性拉伸下，細胞的跳動週期變長。加入釓之後我們發現，拉伸對細胞跳動週期的影響小很多。除此之外，本實驗抑制了纖維母細胞的增長，發現在低密度纖維母細胞的情況下，跳動週期以及心律變異度明顯的高於高密度纖維母細胞的心臟細胞。

Traditionally, it is believed that cardiac dynamics is controlled only by electrical activities. However, it is known that mechanical forces can also activate mechanical sensitive channels in the cell which can bring changes in electrical activity. The purpose of our experiment is to understand whether mechanical forces affect the cardiac beating dynamics. We also want to understand whether the number of fibroblasts which is mechanical sensitive affect the beating dynamics. Cardiomyocytes were dissected from rat embryos and seeded on a deformable silicon chamber in which stretching can be realized by elongation. Two kinds of stretching were used: (1) Periodic stretching at 1 Hz for 2 to 4 hours and the dynamics are observed by calcium imaging, (2) Sustained stretching for 5 minutes and the dynamics are observed by bright field microscopy. The stretch-activated ion channels (SACs) blocker, Gadolinium (Gd3+), was used in our experiment to investigate its effect on the beating dynamics. Under periodic stretching, we found that the frequency of cardiomyocytes can almost be synchronized by the stretching frequency. As for sustained stretching, we found that the heart rate decreases during stretching. After treating with $Gd^{3+}$, the difference of inter-beat interval (IBI) between stretched and non-stretched samples significantly decreases. In addition, we also investigated the beating of cardiac culture in the presence of high and low density fibroblasts. As for low density of fibroblasts, the IBI and heart rate variability is higher than those in the high density one.

摘要  i
Abstract  iii
致謝  v
List of Figures  xi
List of Tables  xvii
Symbols  xix
Chapter 1 Introduction  1
1.1 Objective  1
1.2 The function and structure of the heart  2
1.3 Conduction system  3
1.3.1 SA node  3
1.3.2 Conduction  4
1.3.3 The action potential of cardiomyocyte cell  6
1.3.4 Excitation-contraction(EC) coupling  8
1.3.5 The membrane potential of fibroblast  9
1.3.6 Gap junction  11
1.4 Calcium imaging   11
1.5 Heart rate variability  12
1.6 Previous work on effect of stretching  13
1.6.1 Inward current through stretch-activated channels  14
1.6.2 Morphology changes  15
1.6.3 Increase in Ca2+ spark rate  16
1.6.4 Gap junction conductance  17
1.7 Goal of this work  17
Chapter 2 Method and Experimental Setup  19
2.1 Overview  19
2.2 Sample preparation  20
2.3 Optical system  21
2.3.1 Fluorescent optics system   21
2.3.2 Phase contrast optics system  23
2.4 Stretching system  24
2.4.1 Setup  24
2.4.2 Periodic stretching  27
2.4.3 Sustained stretching  28
2.4.4 Our works  28
2.5 Image Analysis  29
2.5.1 Calcium imaging  29
2.5.2 Bright field image  30
Chapter 3 Results  33
3.1 Overview  33
3.2 Result of periodic stretching (E1)  34
3.2.1 Periodic stretching increase the beating  34
3.2.2 Increasing of reentrant propagation frequency after stretching  34
3.3 Result of sustained stretching  35
3.3.1 Stretching effects on IBI and HRV (E2)  37
3.3.2 Effect of Gadolinium (E3)  38
3.4 Effects on different densities of fibroblasts (E4,E5)  42
Chapter 4 Discussions  49
4.1 Effects of stretching on cardiac dynamics  49
4.1.1 The variation of IBI and HRV on cardiac culture  49
4.1.2 Synchronization between every cluster  50
4.1.3 Effects of different density of fibroblast  50
4.1.4 Effects of SACs blocker   Gadolinium(Gd3+)  51
4.2 Mechanism of stretching on cardiac culture  51
4.2.1 Activities of ion channels  51
4.2.2 Activities of fibroblasts  52
4.3 Future work and improvement  53
4.3.1 Stretchable microelectrode array  53
4.3.2 Immunostaining  53
4.3.3 Compressing on cardiac culture  53
Appendix A  55
A.1 Culture medium preparation  55
A.2 Coating preparation  55
A.2.1 Fibronectin  55
A.2.2 Coating procedure  55
A.3 Dissection  55
A.3.1 Preparation  55
A.3.2 Tissue fragments preparation  56
A.3.3 Digestion  56
A.3.4 Cell suspension preparation  57
A.3.5 Pre-plating (removing the fibroblast)  57
A.3.6 Cell counting / sample plating  57
Appendix B  59
B.1 Tyrode solution (400 mL)  59
B.1.1 Stock solution  59
B.1.2 Working solution  60
B.2 Fluo-8 AM loading stock solution  60
B.3 Staining protocol for cardiac culture is seeded on chamber  60
Appendix C  61
References  63

                                

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