血管組織在動脈硬化過程中的超微結構及生物力學變化研究

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研究生：	暢茁 Chang, Zhuo
論文名稱：	血管組織在動脈硬化過程中的超微結構及生物力學變化研究 Nanoscale Characterisation of Arterial Stiffening
指導教授：	陳伯宇 Chen, Po-Yu Riaz Akhtar Riaz, Akhtar Paolo Paoletti Paolo, Paoletti
口試委員:	莊永仁 Chuang, Yung-Jen 張書偉 Chang, Shu-Wei Pete Twigg Pete, Twigg David Martin David, Martin
學位類別：	博士 Doctor
系所名稱：	教務處 - 跨院國際博士班學位學程 International Intercollegiate PhD Program
論文出版年：	2018
畢業學年度：	106
語文別：	英文
論文頁數：	245
中文關鍵詞：	動脈硬化、生物力學
外文關鍵詞：	Arterial stiffening, Biomechanics
相關次數：	點閱：2 下載：0
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動脈硬化作為一種與年齡相關的老化過程與心腦血管疾病有著密不可分的聯繫。儘管有關動脈硬化的研究在臨床上已經可以檢測，但是對於其形成過程中血管組織結構與生物力學性質的認識卻十分匱乏。該問題主要是由於傳統力學性質研究技術的限制造成的。為了突破該限制，一種全新的納米級別分析結構及力學性質的技術，PeakForce納米力學研究技術 (PeakForce QNM) 在該論文中被使用。首先該技術被用於研究斑馬魚脊柱骨的微結構及其生物力學性質隨著年齡增長發生的變化。通過斑馬魚的模型，驗證了該技術在研究小尺度生物樣品和結構的可行性。為下一步研究人體血管及其硬化相關的變化鋪平了道路。

該研究使用人體胸腔內動脈作為研究模型，探索動脈硬化過程中血管的結構及力學性質變化。其中，主要研究動脈的中膜與外膜的變化。通過使用PeakForce QNM技術，動脈組織的微觀結構分別在氣象和液相中被研究，其中的膠原纖維結構變化也被研究。研究發現，血管各層的生物力學性質，外膜的膠原纖維形態與其動脈硬化程度相關。此外，蛋白質組學實驗發現了與動脈硬化相關的蛋白也與該實驗結果相關，解釋了血管微觀結構，納米生物力學以及宏觀動脈硬化程度的相關性。該研究可悲應用於早期診斷血管疾病。

Arterial stiffening as part of the natural ageing process is strongly linked to cardiovascular risk. Although arterial stiffening is routinely measured in vivo, little is known about how localised changes in artery structure and biomechanics contribute to in vivo arterial stiffening. This is mainly due to the limitation of the conventional mechanical testing methods.

To circumvent this challenge, a novel nano-scale structural and mechanical characterisation technique, known as PeakForce Quantitative Nanomechanical Mapping (QNM) technique, was developed in a zebrafish model. Using the zebrafish vertebral column, the utility of the PeakForce QNM for probing small-scale biological samples and structures was validated, which paved the way to probe human artery and investigate the localised alterations in artery structure in vitro with arterial stiffening.

Human internal mammary artery (IMA) was used as a model vessel for understanding the development of arterial stiffening in this thesis. This thesis focuses on the role of the tunica media and the outmost layer, the tunica adventitia, in arterial stiffening. Using the PeakFoce QNM, the hydrated and dehydrated arterial sections were tested that provided data on nano-scale changes in collagen fibril structure and mechanical properties in the hydrated media, dehydrated media and adventitia and showed how they related to in vivo stiffness measurements in the vascular system. The indentation depth for AFM measurement on the IMA tissues of 5 µm thickness were controlled at 20 nm and 5 nm in liquid and ambient conditions respectively and thus the indentation depth/tissue thickness ratio was 0.4% and 0.1% for the hydrated and dehydrated samples respectively. Furthermore, integrating the findings in this thesis with the proteome analysis data, the localised alterations in the collagen and ultrastructure were explained, and the in vivo arterial stiffening, nanomechanical and structural changes in artery biopsy samples were linked. This approach could be used to develop new diagnostic methods for vascular disease.

Contents
Chapter 1. Introduction
1 Motivation    2
2 Aims and Objectives    4
References    5

Chapter 2. Literature review
1 CVDs    8
1.1 Background    8
1.2 CVD in the UK, Europe and worldwide    9
2 Arterial stiffening    11
2.1 Background    11
2.2 Relationship between arterial stiffening and CVD    11
2.3 Assessment of arterial stiffening with PWV    12
3 Arterial structure and classification    13
3.1 Arterial structure    13
3.1.1 Tunica intima    13
3.1.2 Tunica media    15
3.1.3 Tunica adventitia    17
3.1.4 Collagen    17
3.1.5 Elastin    20
3.1.6 SMCs    21
3.1.7 Proteoglycans and small leucine-rich proteoglycans (SLRPs)    21
3.2 Classification of arteries    22
3.2.1 Elastic arteries    23
3.2.2 Muscular arteries    25
3.2.3 Transitional arteries    25
4 Biomechanical properties of the artery wall    26
4.1 Macroscale mechanical testing methods and tensile testing    29
4.1.1 Macroscale mechanical testing methods    29
4.1.2 Tensile testing    29
4.2 Microscale - nanoindentation    30
4.3 Nanoscale - AFM    32
5 Alterations in arterial composition and biomechanics with age-related arterial stiffening    33
5.1 Alterations in collagen    33
5.2 Alterations in elastin    34
5.3 Alterations in SMCs    35
5.4 Alterations in proteoglycans and SLRPs    35
5.4.1 Influences on collagen formation and organisation    36
5.4.2 Influence on elastin formation and organisation    36
5.4.3 Influence on SMCs    37
6 AFM    40
6.1 Biological application of AFM    40
6.2 Principles of AFM    41
6.2.1 AFM instrumentation    41
6.2.2 Working modes    42
6.3 Characterisation of mechanical properties with AFM    44
6.3.1 Force curves    44
6.3.2 Contact models    45
6.3.3 PeakForce QNM    49
6.3.4 Nanoscale imaging and mechanical characterisation and its application to  biomedicine      56
7 Summary    57
References    59

Chapter 3. Technique development for nanoscale characterisation of biological samples in a zebrafish model
Abstract    81
1 Introduction    82
1.1 Zebrafish as a model for human diseases    82
1.1.1 The use of zebrafish in cardiovascular research    82
1.1.2 Rationale for focussing on the zebrafish vertebral column    83
1.2 Zebrafish as a model to study bone maturation: Nanoscale structural and mechanical characterisation of age-related changes in the zebrafish vertebral column    84
2. Materials and methods    86
2.1 Sample information    86
2.2 Sample preparation    87
2.3 Hematoxylin and eosin staining    87
2.4 Scanning electron microscopy (SEM)    88
2.5 Energy-dispersive X-ray spectroscopy (EDX) analysis    88
2.6 PeakForce QNM AFM    89
2.7 Statistical analysis    91
3 Results    92
3.1 Histological analysis    92
3.2 Bone ultrastructure    94
3.3 Chemical compositions in the bone    98
3.4 Nanoscale mechanical properties    99
3.5 Principle component analysis    101
4 Discussion    102
4.1 Zebrafish as a bone ageing model    103
4.2 Wall thickness    104
4.3 Ultrastructural properties of zebrafish bone    104
4.4 Ca/P ratio    105
4.5 Elastic properties    106
5 Limitations    108
6 Conclusions    110
7 Relevance of work to arterial stiffening    111
References    112

Chapter 4. Nanomechanics and ultrastructure of the IMA adventitia in patients with low and high PWV
Abstract    122
1 Introduction    123
2 Materials and Methods    125
2.1 Clinical characterisation    125
2.2 PeakForce QNM characterisation    126
2.3 Collagen fibril analysis    129
2.4 Histology    131
2.5 Integration of quantitative proteomics, nanomechanical data and patient metadata    132
2.6 Statistical methods    132
3 Results    135
3.1 Histological analysis    135
3.2 Nanomechanical properties    137
3.3 Morphology of adventitial collagen fibrils    140
3.4 Principal component analysis    144
4 Discussion    147
4.1 Nanomechanics of the adventitia    148
4.2 Collagen fibril diameter    149
4.3 Collagen fibril D-period    150
5 Limitations    152
6 Conclusions    153
References    155

Chapter 5. Characterisation of ultrastructural changes in the medial layer of the IMA in patients with arterial stiffening
Abstract    163
1 Introduction    164
2 Materials and Methods    165
2.1 PeakForce QNM study in ambient condition    165
2.2 PeakForce QNM study in a fluid environment    166
2.3 Characterisation of collagen fibril size    167
2.4 Integration of quantitative proteomics, nanomechanical data and patient metadata    168
2.5 Statistical methods    168
3. Results    169
3.1 Collagen fibrils in the hydrated and dehydrated medial layer    169
3.3 Nanomechanical properties of dehydrated media in ambient condition    173
3.4 Nanomechanical properties of hydrated media in a fluid environment    175
3.5 Correlation analysis    176
3.6 Principle component analysis    179
4 Discussion    180
4.1 Collagen fibrils in the IMA    181
4.2 Mechanical properties of the hydrated and dehydrated IMAs    183
4.3 Role of SLRPs in the IMA mechanical properties    189
5. Conclusions    190
References    191

Chapter 6. Discussion and Limitations
1 Discussion    201
1.1 Nanomechanical mapping by AFM PeakForce QNM    201
1.2 Characterisation of small-scale biological structures and tissue ECM components    203
1.3 Stiffening of the human IMA    204
2 Limitations    207
2.1 PeakForce QNM technique limitations    207
2.3 Sample limitations    208
2.3.1 Zebrafish model    208
2.3.2 Human IMA    208
2.4 Limitations with the experimental approach    209
2.4.1 Zebrafish vertebral column study    209
2.4.2 Human IMA study    209
2.4.3 Nanomechanical data analysis……………………………………………  …..209
References    211

Chapter 7. Conclusions and Future work
1 Conclusions    219
2 Overall summary    222
3 Future work    223
3.1 Nanomechanical mapping in a physiological environment    223
3.2 Demineralisation for revealing the collagen fibrils in zebrafish skeleton    223
3.3 Characterisation of IMA tunica intima    224
3.4 Quantitative assessment of the collagen and elastin content in different layers    224
3.5 Layer-specific distribution of the SLRPs and other ECM remodelling associated proteins    224
Reference    225
Appendix A-G………………………………………………………………………………218

                                

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