臺灣博碩士論文加值系統

上皮細胞組成後生動物絕大部分的組織，也進行許多重要的生理反應，像是物理性的保護、選擇性的分泌與吸收。Tight junction是上皮細胞間緊密相連的帶狀屏障，使細胞膜極化為apical、lateral、basal三個區域。Tight junction形成在靠近apical表面的細胞間隙，無法讓膜蛋白通過的性質使得上皮細胞高度極化。肌動蛋白(Actin)連接在Tight junction上的銜接蛋白上，使其結構成為了解細胞力學重要的一環。我們發現Tight junction在MDCK細胞(Madin Darby Canine Kidney)中形成彎曲的構造，且同時表現在2D緊密的transwell還有matrigel上的3D cyst中。我們發現使用Y27632, blebbistatin還有ML-7來影響MDCK肌動蛋白-肌球蛋白(actin-myosin)的收縮，可以減少Tight junction的彎曲程度以及細胞高度和apical面積。另外，使用雷射將單一細胞的肌動蛋白燒掉也可以增加其apical面積以及減少Tight junction的彎曲程度。使用3D影像處理可以量化Tight junction的彎曲程度，量化的資訊可以反映apical和lateral的表面張力，用Surface Evolver進行理論模擬也成功得到類似的預測。使用細胞本身的結構來推導出細胞間的拉力是非侵入式的方法，得到的資訊可以用來檢視現有的理論模型。

Epithelium comprises the majority of metazoan structures and perform important physiological functions such as protection barrier, secretion, and selective absorption. They are highly polarized and the plasma membranes are separated into apical, lateral, and basal sides. Tight junctions form a continuous belt at the sub-apical location at the borders of two cells as a fence function to maintain the polarity of membrane proteins and seal the paracellular space between cells. The tight junctions are linked to actin cytoskeleton through adaptor proteins. We found that the tight junctions form tortuous structure as Madin Darby Canine Kidney (MDCK) cells grow into higher confluency on a 2D transwell or as a cyst in 3D matrigel. When we perturbed actin-myosin contractility of MDCK cells by small molecules Y27632, blebbistatin, and ML-7, the tight junctions become less tortuous, and cell shape changes in terms of height and the apical area. Moreover, when actin filaments at the apical surface were ablated, we observed an expansion in apical area and a change in tortuosity of tight junction. We developed 3D image analysis to quantify the tortuosity of tight junctions and proposed that the morphological change of tight junctions can be indication of apical constriction force and cell-cell tension. By constructing a simple theoretical model by surface evolver to explain the morphology of tight junction affected by the interplay between apical, and lateral tension. Using the endogenous cellular structure for quantify intercellular force is non-perturbative and the gained knowledge can be used to test current theoretical models which explains the epithelial cell shapes based on basal, lateral, and apical tensions.

Contents
中文摘要 ii
Abstract iii
Acknowledgement v
Contents vi
List of Tables x
Chapter 1 Introduction 1
1.1 Epithelium 1
2
1.2 Cell-cell adhesions 2
1.3 Actomyosin network 3
1.4 Tissue morphogenesis and cystogenesis 4
Chapter 2 Methodology 7
2.1 Cell culture 7
2.2 Culturing MDCK cysts 7
2.3 Culturing polarized MDCK cells in 2D transwell 9
2.4 SiR-actin label 10
2.5 Actin-myosin contractility perturbation 10
2.6 Laser ablation 11
2.7 Immunostain of MDCK cysts 11
2.8 Image acquisition 12
2.9 Image processing and segmentation 13
2.10 Simulating curves for curvature analysis 14
2.11 Curvature analysis 15
2.12 Cell shape analysis 15
2.13 Error estimation 16
2.14 Surface evolver 16
Chapter 3 Results and Discussion 22
3.1 Accuracy of Rc calculation based on simulated curves 22
3.2 Transwell analysis 22
3.3 MDCK cyst analysis 23
3.3.1 Day to day observation 23
3.3.2 Y-27632 treatment 24
3.3.3 Blebbistatin treatment 25
3.3.4 ML-7 treatment 26
3.3.5 Comparison of Y-27632, blebbistatin, and ML-7 27
3.4 Laser ablation 27
3.5 Error estimation 28
3.6 Surface evolver 28
Chapter 4 Conclusion 49
References 51

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