臺灣博碩士論文加值系統

細胞應力可由細胞內部或外部的因子產生，並能調控細胞重要的生理功能，例如生長、分裂、增生、分化與移動等；而細胞與細胞之間應力的感受和傳遞，則是透過細胞間黏著點與細胞骨架和肌動蛋白的協調，除了可將構成力量的物理訊號轉變成生物化學訊號，細胞間黏力的變化更是調節細胞生理狀態的首要因素。鈣敏黏性蛋白(Cadherin) 為ㄧ主要調控細胞間黏力的黏性蛋白質分子，其形成的黏性結構廣泛存在於組織與細胞中，如神經元的突觸，上皮細胞間黏著點等；但是，細胞如何藉由鈣敏黏性蛋白與其連結的相關分子和細胞骨骼來感受和傳遞應力，目前的研究還尚無定論。

本論文以控制細胞的生長環境、形狀與基質硬度出發，試圖建立新的細胞間黏力量測平台，由此研究鈣敏黏性蛋白與其相關分子如何影響細胞間應力的傳遞。我們選用的黏性微圖陣為一成對之等腰三角形，其間隙大小為8微米，用以調控兩顆細胞的生長形狀與受力方向；牽引力顯微鏡(Traction Force Microscopy)則是用於量測細胞間的作用力的大小；至於研究所使用的細胞，則為大量表達上皮性鈣敏黏性蛋白(E-Cadherin)的小鼠乳腺上皮細胞 (EpH4)，與利用CRISPR技術剔除與鈣敏黏性蛋白和相關基因的細胞株。
本研究結果發現；利用三角成對的黏性微陣圖列，並改變培養液鈣離子的濃度，細胞間黏著力可以成功地藉由螢光粒子的位移被量測(μN範圍)；結合基因剔除可能與力量傳導相關的分子如p120連環蛋白與抗肌萎蛋白，我們則發現: 1.在改變細胞外鈣離子濃度前，平均力量會因基因剔除而減少；2.在加回鈣離子的回復過程中的，EpH4野生型細胞具有最佳回復黏性的能力； 3.剔除抗肌萎蛋白則顯助減弱黏力回復，而剔除p120連環蛋白則完全抑制黏力恢復； 4.剔除抗肌萎蛋白會造成胞膜穩定性降低。總結本系列實驗，首先本團隊巧妙連結黏性微圖陣與牽引力顯微鏡，使之可合理測量鈣敏黏性蛋白質媒介之細胞間黏力；再者，利用此獨特的方法我們發現，p120連環蛋白與抗肌萎蛋白皆在細胞間黏力的產生與傳遞的過程中扮演負向調節的角色。

Cellular mechanical forces can be produced by intrinsic/extrinsic factors; these physical cues regulate a wide array of cellular functions, such as cell growth, division, proliferation, differentiation, and migration. It is believed that mechanical forces generated by cell-cell interaction are able to transmit to the interior of cell through the coordination of cortical actomyosin network; within the process, the physical signals can be transmitted to chemical ones. Prominent among other adhesive membrane receptors, Cadherins are prototypical calcium-dependent adhesive molecules able to construct adhesive interfaces between cells, such as Adherens Junctions in epithelial cells and Synapses neurons. Cadherin can generate remarkable forces to regulate intercellular adhesion. However, the mechanistic steps of mechanotransduction in Cadherin-mediated adhesion remain very controversial.
We are interested in understanding how Cadherin protein complexes enable force generation and transmission at cell-cell adhesion in the initial stage of intercellular adhesion. For providing a better control of time, space and substrate stiffness, in this study, a combination of protein micropattern and traction force microscopy (TFM) is attempted to utilize in our experiments. Pair micropattern with triangle forms confines cell spreading area and the gaps in pair is 8 microns applied for monitoring the force that cell pairs generated, measured by TFM. Moreover, in addition of wildtype (WT) mouse breast epithelial cell line (EpH4), clones obtained from epithelial cells undergone genome editing (CRISPR) are used to score the importance for known components of Cadherin complexes in force generation.
By using aforementioned methodology to investigate cell-cell adhesion and measure the strengths conferred to cell by E-Cadherin interactions in comparison of WT, knockout (KO) of p120 catenin (p120-/-) and KO of Utrophin gene (Utrn-/-) cells respectively, we found that : 1) gene KO reduces average forces obtained by TFM; 2) after calcium switch, WT cells have the best capability to recover E-Cadherin-mediated cell-cell adhesion; 3) Utrn-/- cells display a reduced recovery of cell-cell adhesion whereas this recovery in p120-/- cells is abolished; 4) Utrn-/- cells display a significant instability at plasma membrane activities. Taken together, we believe that these results show both p120 catenin and Utrophin play a negative regulatory role in cadherin-mediated intercellular adhesion and that the effect of Utrophin may be resulted from the instability of plasma membrane. Our combinatory mechanobiological method could provide deep insights into understanding the biophysical principle governing mechanotransduction of Cadherin biology.

Content
Acknowledgement (致謝)..................................Ⅰ
Chinese Abstract (中文摘要).............................Ⅱ
English Abstract.......................................Ⅳ
Content................................................Ⅵ
List of Tables........................................Ⅷ
List of Figures........................................Ⅸ
List of Abbreviations..................................Ⅹ
1. Introduction...................................1
1.1 Force measurement between cell pairs...........2
1.2 Cadherin molecular complex.....................3
1.3 Force measurement for cell-cell adhesion by TFM.4
1.4 Application of micropatterning in cell biology.5
1.5 Objectives of research.........................6
2. Material and Method............................7
2.1 Designing of photomask.......................7
2.2 Coverslip silanization.......................7
2.3 Preparation of fibronectin solution..........8
2.4 Preparation of PLL-PEG solution..............8
2.5 Preparation of polyacrylamide gels...........8
2.6 Fabrication of micropatterned PAA gels......11
2.6.1 Photomask Activation..........................11
2.6.2 Passivation...................................11
2.6.3 UV treatment and protein coating..............12
2.6.4 Transfer on acrylamide gel....................12
2.7 Cell culture................................15
2.7.1 Cell line for the experiment..................16
2.8 Live cell imaging...........................17
2.9 Traction force microscopy and image analysis.17
3. Results.......................................18
3.1 Micropattern on the PAA gels................18
3.2 Culturing cell on the pair micropattern.....20
3.3 Variations of cell-cell adhesive strength in the process of calcium-switch.........................21
3.4 Continuous time measurement of cell-cell adhesive strength.....................................24
3.5 Normalized Force comparison.................30
4. Discussion....................................31
5. Conclusions...................................33
6. Reference.....................................34

List of Tables
Table 1 Selection of polyacrylamide gel stiffness......10

List of Figures
Figure 1 Cadherin Molecular Complex in cell-cell adhesion................................................4
Figure 2 Schematic diagram of micropatterning on PAA gel fabrication............................................14
Figure 3 Micropattern on the PAA gels..................19
Figure 4 Cell cultured on the pair micropattern........20
Figure 5 Cell-cell adhesion force variation in restoring process................................................23
Figure 6 Eph4WT cell-cell adhesion forces in continuous time measurement.......................................25
Figure 7 Eph4U3 cell-cell adhesion forces in continuous time measurement.......................................26
Figure 8 The different membrane behavior between Eph4WT and Eph4U3 in DIC image................................27
Figure 9 Eph4C2 cell-cell adhesion forces in continuous time measurement.......................................28
Figure 10 Cell-cell adhesion force results of three cell lines..................................................29
Figure 11 Linear regression analysis results in restoring process................................................29
Figure 12 Comparison of normalized cell-cell adhesion forces.................................................30

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