臺灣博碩士論文加值系統

細胞應力可由胞外或胞內的多種因子產生，並從而調控許多細胞生理過程，如：生長、分化及遷移力。細胞間的應力可以透過細胞與細胞間彼此相連，透過肌動球蛋白網絡與應力感應分子的協調，將物理訊號轉變為化學訊號再傳遞至細胞內部。然而，目前尚無較直接的方法來量測在二維的基質上細胞間黏力。因此，藉由結合已建立的生物物理與細胞生物學的方法，開發一套準確的細胞間應力量測技術是當務之急。
上皮鈣敏黏性蛋白質(E-Cadherin)是一種調控細胞間黏力的主要分子，他可以透過β-連環蛋白和α-連環蛋白快速地與纖維性肌動蛋白連接，產生強力的細胞間黏力。但是，細胞如何藉由鈣敏黏性蛋白與其連結的相關分子來感受和傳遞應力，目前的研究還尚無定論。因此，本篇研究我們試圖開發一種新的方式來測量貼附形細胞間的應力。我們將牽引力顯微鏡與黏性微陣圖列結合，透過控制細胞形狀的技術，結合牽引力的運算來推算細胞間的應力。研究所選用的細胞為小鼠上皮乳腺細胞(EpH4)，具有表現大量上皮黏性蛋白的特性，並可貼附在成對的黏性微陣圖列上。實驗時則利用迅速移除與回復鈣離子濃度的過程來模擬改敏黏性蛋白重組的狀態，再搭配利用CRISPR技術剔除與鈣敏黏性蛋白和相關基因的細胞株，觀察由鈣敏黏性蛋白所調控的細胞應力變化。
本論文研究結果發現，利用成對的三角形黏性為陣圖列，並調控培養液中鈣離子的濃度，細胞內的應力透過基質中螢光粒子位移大小來計算長軸力而作為細胞間的黏力。我們比較過經基因剔除與鈣敏黏性蛋白相關並參與調控應力傳遞分子，如：p120連環蛋白、抗肌萎蛋白、α-連環蛋白等等細胞後，我們發現1.在重組細胞黏力時，前30分鐘內的細胞應力是來自於細胞貼附基質的胞膜擴張 2. 野生型的細胞具有較強的細胞黏性，重建細胞黏性所需時間也較短 3. 在鈣離子轉換前，細胞間的力量會因不同基因被剔除而有相對應減少，當中又屬剔除p120連環蛋白的細胞力量最小 4. 細胞剔除鈣敏黏性蛋白基因與剔除α-連環蛋白兩株細胞在重組細胞間應力時，所需時間較少 5. 細胞剔除p120連環蛋白與抗萎蛋白基因的兩株細胞於建構細胞黏力的過程中則有相似的表現，重建時間也較久。綜觀本研究，我們成功地結合牽引力顯微鏡與黏性為陣圖列，使之得以觀察以鈣敏黏性蛋白為媒介還調控的細胞間的應力變化；另外我們重新定義了p120連環蛋白、α-連環蛋白與抗肌萎蛋白在黏性介面重組的角色。

Cellular mechanical forces can be generated intrinsically and extrinsically; these physical cues regulate a wide variety of critical cellular functions such as cell proliferation, differentiation, and migration. Mechanical forces generated by cell-cell interaction can transmit to the interior of the cell through the coordination of cortical actomyosin networks. Following that, physical signals can be converted to chemical ones through mechanotransducing process. However, force conferred by the adhesive receptors to cells measurements methods on 2D substrate is not straightforward. Thus it is an urgent need to develop functional assay able to measure the intercellular strengths by known biophysical model and cell biology tools.

E-Cadherins (E-Cdh) are prototypical adhesive molecules able to construct strong adhesive interfaces between cells by rapid assembly of E-Cdh complex with association of cytoskeleton actin. Currently E-Cdh complexes comprised of β-Catenin and α-Catenin are suggested to associate with cortical F-actin in a dynamic manner at nascent cell-cell contact; however, the mechanistic steps of mechanotransduction in E-Cdh-mediated cell adhesion remain elusive. In this study, we attempt to establish a novel protocol for measuring intercellular forces generated by two adhering cells on 2D flexible substrate with a precise temporal and spatial control. A combination of protein micropattern and traction force microscopy (TFM) is therefore developed. EpH4 cells of mouse breast epithelial origin are allowed to form the cell doublets on micropattern and then calcium switch (CS) is proceeded to initiate the E-Cdh-mediated cell adhesion. A series of CRISPR clones targeted primarily on E-Cdh and its associated proteins are prepared to decipher the novel mechanotransduction pathway of E-Cdh.

By using the aforementioned methodology, we investigate cell-cell adhesion strength mediated by E-Cadherin interaction in comparison of WT, knock out of E-Cadherin (E-Cdh-/-), p120-Catenin (p120-/-), Utrophin (Utrn-/-) and α-Catenin (α-cat-/-) cells respectively, we found that: 1) Force we measured in the first 30 min after CS is primarily generated by cell spreading, 2) WT cells generated the highest long axis force (852.34±6.9 nN) and the better capability to recover in E-Cadherin-mediated cell-cell adhesion after CS (56.52 ± 14.6 mins); 3) the average force of all the gene knock out cells are reduced, especially p120-/- cells have the weakest force (417.8 ± 8.1 nN) before CS; 4) E-Cdh-/- cells and α-cat-/- cells only need short time to let the force being balanced after CS while p120-/- cells and Utrn-/- cells require longer time (4.05 ± 0.6 hrs; 4.67 ± 1.4 hrs) to recover; 5) During the processes of rebuilding nascent E-Cadherin–mediated adhesion, p120-Catenin and Utrophin display similar maximal long-axis force. We believe that our combinatory force measurement method provides better insights on the machanotransduction strength of Cadherin-mediated cell adhesion.

Content
致謝 I
中文摘要 III
English Abstract V
List of Tables IX
List of Figures X
List of Abbreviations XI
1. Introduction 1
1.1 Force Transmission in Cells 1
1.1.1 Mechanosensing molecules 2
1.1.2 E-Cadherin adhesive complex 3
1.1.3 Utrophin as a plausible novel member of Cadherin complex 5
1.2 Force Measurement Methods 6
1.3 Application of Micropattern in Cell Biology 8
1.4 Objective of research 8
2. Materials and Methods 9
2.1 Preparation of micropatterning 9
2.1.1 Design of a photomask 9
2.1.2 Coverslip silanization 9
2.1.3 Preparation of poly-L-Lysine-PEG solution 10
2.1.4 Preparation of fibronectin solution 11
2.1.5 Preparation of polyacrylamide gel 11
2.2 Fabrication of pair micropattern on 9 kPa polyacrylamide gel 13
2.2.1 Coverslips surface activation 13
2.2.2 Passivation by PLL-PEG 13
2.2.3 Deep UV illumination 14
2.2.4 Extracellular protein (fibronectin) coating 14
2.2.5 Transfer Micropattern on polyacrylamide gel 14
2.3 Cell culture 16
2.3.1 Cell lines for the Experiment 17
2.4 Live cell imaging 19
2.5 Traction force measurement and image analysis 21
3. Results 22
3.1 Micropattern on 9 kPa polyacrylamide gels 22
3.2 Culture cells on micropatterning polyacrylamide gels 23
3.3 Various cell-cell adhesive strengths from CRISPR clones 24
3.4 Comparison of long axis force in EpH4 CRISPR clones 26
3.5 Time required for reaching to force balance in EpH4 cells 28
4. Discussion 30
5. Conclusion 37
References 38


List of Tables
Table 1. Selection of polyacrylamide gel stiffness. 12
Table 2. Comparison of long axis forces in different EpH4 cell lines 27
Table 3. Time required for the intercellular forces to reach the equilibrium after CS in different EpH4 cell lines. 29


List of Figures
Figure 1. E-Cadherin adhesive complex at cell-cell contact.. 4
Figure 2. Localization of Utrophin at cell-cell contact is dependent on p120-Catenin.. 6
Figure 3. Production of coverslip silanization 10
Figure 4. Schematic diagram of micropatterning on polyacrylamide gel fabrication. 16
Figure 5. Micropattern on 9 kPa Polyacrylamige gels. 22
Figure 6. EpH4WT seeding on PAA gels with micropattern. 23
Figure 7. Balance of cell-substrate traction forces and cell-cell adhesion forces. 25
Figure 8. Long axis forces in continuous time measurement. 27
Figure 9. Different recovery slope in restoring process. 28
Figure 10. Real time monitoring the barrier function recovery after CS by ECIS. 36

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