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研究生：	柯奕恩 Mhar Ian Cua Estayan
論文名稱：	The Effects of Sustain Stretching and Compression in the Inter-beat Interval and Beat Rate Variability of Embryonic Cardiomyocytes
指導教授：	陳志強 Chi-Keung Chan 黎璧賢 Pik-Yin Lai
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：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	105
中文關鍵詞：	心率、壓縮
外文關鍵詞：	Mechanical Stretching, Beat Rate Variability, Inter-beat Interval
相關次數：	點閱：16 下載：0
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許多的觀察指出心肺復甦術是在心臟上施加機械應力而使得心臟重新跳動。構成心臟的細胞主要有心肌細胞以及纖維母細胞兩種細胞都具有感應機械應力的離子通道並且可以被釓所抑制這項研究的目的是想要觀察在不同的密度的纖維母細胞的心肌細胞培養中，機械應力是否影響心肌細胞的動態反應。為了更詳細瞭解機械應力對心臟的動態的影響，我們設計了兩種機械應力的刺激方式: 1.) 持續性拉伸以及 2.)持續性壓縮。我們記錄了心肌細胞跳動的影響並且計算了的脈衝間隔以及心律變異。我們的研究發現機械應力對於有高濃度的纖維母細胞的心肌培養之脈衝間隔以及心律變異沒有顯著性影響，加入釓亦無影響。在攝氏27度的時候，機械應力可以影響低濃度的纖維母細胞的心肌培養之脈衝間隔以及心律變異。

Mechanical stress on the heart has been observed to retrieve heart beating by means of performing cardiopulmonary resuscitation. Cardiomyocyte and Fibroblast are cells that comprises the heart. Both cells have stretch activated ion channels which can be blocked by Gadolinium. The purpose of this experiment is to observe if mechanical stress affects the cardiac dynamics in cellular level and to investigate the different densities of fibroblast. To understand the cardiac dynamics under mechanical stress, we designed two types of mechanical stimulation: 1.) Sustain Stretching and 2.) Compression. Percentage change in the Inter-Beat Interval (IBI) and Beat Rate Variability (BRV) were computed. The result of this experiment shows that mechanical stretch does not affect the IBI and BRV for samples with high number of fibroblast. Gadolinium does not affect IBI and BRV when drug is added. Mechanical stress at 27˚C affects BRV and mechanical stress of low density fibroblast affects the IBI and BRV.

Introduction…………………………………………………………………...1
1 Overview……………………………………………………….....1
2 Anatomy and Physiology of the Heart……………………………1
3 Cardiomyocytes, Fibroblast and Gap Junction……………………3
4 Synchronization of Cardiomyocytes………………………………8
5 Stretch Activated Ion Channels…………………………………..12
6 Temperature Effect……………………………………………….14
7 Previous Research on the Effects of Mechanical Stress………….14
7.1 Mechanically Induced Potential of Fibroblast……………..14
7.2 Stretching can induce Calcium Transient………………….15
7.3 Sino-Atrial (SA) node stretching reduced Heart Rate Variability………………………………………………….16
7.4 Cyclical Stretching maturates cardiomyocyte……………..17
7.5 Cyclical Mechanical Stretch Induced Vascular Endothelial Growth Factor……..………………………………………18
7.6 Cyclical Stretching affects Cardiomyocyte Cellular Organization……………………………………………….19
7.7 Effects of Periodic and Sustain Stretching in IBI and HRV.20
8 The Objective of the Research……………………………………21
Methodology…………………………………………………………………...22
1 Overview………………………………………………………….23
2 Preparation of Samples……………………………………...……23
2.A Cardiomyocytes and Fibroblast Preparation……………….23
2.B Decreasing the amount of Fibroblast………………………24
2.C Immunocytochemistry and Cell Counting…………………24
2.D Effects of Gadolinium……………………………………...26
3 The Whole System…………………………………………………..27
3.A The Set Up…………………………………………………27
3.B PID Temperature Controller……………………………….29
4 Calibration and Procedure………………………………………..…32
4.A Procedure for Stretching…………………………………...32
4.B Procedure for Compression and the Module for Compression……………………………………………….33
5 Image Analysis…………………....………………………………35
5.A Background Subtraction……………………………………35
6 Percentage Change of IBI and BRV………………………….…36
7 Statistical Analysis……………………………………………....37
7.1. P-value……………………………………………………..37
7.1.A. Welch’s T-test……………………………………38
Result.................……………………………………………………………….40
1 Overview………………………………………………………….40
2 Time Effect……………………………………………………….40
3 Effects of Mechanical Stress……………………………………...43
3.A. Time Dependence of IBI……………...…………………...43
3.B. Percentage Change in IBI and BRV of stretching are mostly negative for normal samples……………………………………...44
3.C Percentage Change in IBI and BRV of compression are mostly negative for normal samples……………………………...45
3.D Stretching of normal samples does not affect the IBI and BRV………………………………………………………………47
3.E Compression does not affect the IBI and BRV…………….49
4 Effects of Gadolinium…………………………………………….50
4.A 100μM Gadolinium does not affect the IBI and BRV……..51
4.B IBI increases insignificantly when 100μM Gadolinium is added……………………………………………………………...52
4.C Percentage Change in IBI and BRV of stretching and compression with Gadolinium are mostly positive……………….52
4.D Stretching of samples with Gadolinium does not affect the IBI and BRV……………………………………………………...54
4.E Compression of samples with Gadolinium does not affect the IBI and BRV……………………………………………………...55
5 Effects of Low Temperature……………………………………...56
5.A Percentage change of stretching and compression at 27˚C are mostly in the negative region……………………………………..56
5.B Stretching and Compression with Gadolinium reduces the percentage change in IBI…………………………………………58
5.C Stretching at 27˚C does not affect the IBI and but affects BRV………………………………………………………………60
5.D Compression at 27˚C does not affect the IBI but affects BRV………………………………………………………………61
5.E Stretching with Gadolinium at 27˚C does not affect the IBI and BRV…………………………………………………………..62
5.F Compression with Gadolinium at 27˚C does not affect the IBI and BRV…………………………………………………………..63
6 Immunocytochemistry reveals reduction of Fibroblast when treated with Ara-C………………………………………………………..65
7 Low Density Fibroblast compression and stretching……………..65
7.A IBI and BRV of N, F(-) and F(+)…………………………65
7.B Stretching and Compression of F(-) with and without Gadolinium decreases IBI, stretching decreases BRV, and compression increases BRV……………………………………...68
7.C Stretching and Compression of F(-) with and without 100μM Gadolinium does affect the IBI and BRV………………………...71
Discussion, Recommendation and Summary…………………..…................75
1 Discussion……………………………………………..…………….75
1.A Effects of Sustain Stretching and Compression……………76
1.B Effects of Gadolinium……………………………………...76
1.C Effects of Temperature……………………………………..77
1.D Effects of Low Density Fibroblast…………………………77
1.E Comparison of the result to review of related literature……78
2 Recommendation…………………………………………………….79
2.A Cell Type…………………………………………………...79
2.B Increase Number of Samples……………………………….79
2.C Scaffold Cyclical Stretching and Protein Characterization...79
2.D Development of Stretchable Multi-electrode Array………..79
2.E Different Percentage of Stretching…………………………80
3 Summary…………………………………………………………….80
Appendix A…………………………………………………………………….81
Appendix B………………………………………………………...…………..86
Appendix C……………………………………………...……………………..88
Appendix D…………………………………………………………………….89
Appendix E……………...……………………………………………………..90
Bibliography…...………………………………………………………………95
                                

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