鉀氯離子共同通道同質體3 (potassium chloride cotransporter isoform 3, KCC3)是細胞內滲透壓平衡的決定性因子，同時也在腫瘤生物學裡扮演重要的角色。目前它被發現與上皮間葉細胞轉換(epithelial-mesenchymal transition, EMT)的調節相關，亦顯示出KCC3過度表現的SiHa細胞(KCC- overexpressed SiHa Cell)擁有EMT的許多特徵，如拉長了細胞的形狀，降低了上皮細胞的標記(E-cadherin and-catenin)，增強了間葉細胞的標記(vimentin)等。E-cadherin與-catenin為形成細胞與細胞之間連接(cell-cell junction)的其中一類：黏著連接(adherent junction)。而黏著連接形成後提供細胞骨架(cytoskeleton)結合點來增加細胞的韌性，故原生型(wild type SiHa Cell)與KCC3細胞相比，在細胞連接的區域應該會有較大的韌性。
因此我們選擇原子力顯微術(atomic force microscopy, AFM)可以細胞正常生理環境下量測的優點，配合微奈米薄膜理論(thin film theory)來計算不含基材效應的機械特性的能力，以進行細胞連接區域的研究。首先我們在韌性較金屬低的高分子材料，聚二甲基矽氧烷(PDMS)上驗證薄膜理論用確實可以運用在於低韌性的材料，得到修正了基材效應後的楊氏模數。下一步我們在更低韌性的PAA Gel (polyacrylamide gel)上培養細胞，對細胞連接區域進行量測。結果顯示，原生型細胞在細胞連接的區域(3.1 m)比KCC3細胞(1.4 m)擁有較大的厚度，證實原生型細胞擁有較完整的細胞連接。此外原生型細胞在細胞連接的區域(1.5 kPa)有較其它區域更強的韌性(0.6~1.2 kPa)，而EMT細胞(KCC3)則無此現象(1.0~1.2 kPa)。證明了原生型細胞在細胞連接形成後會強化了附近的細胞骨架，而形成穩固的細胞連接。所以以上兩個證據顯示EMT細胞(KCC3)在細胞相鄰區域沒有形成顯著的細胞連接。

The potassium chloride cotransporter isoform 3(KCC3) is a major factor of osmotic homeostasis and plays an emerging role in tumor biology. It is involved in the regulation of epithelial-mesenchymal transition (EMT), a critical cellular event of malignancy. Furthermore, the characteristics of EMT appear in KCC3-overexpressed cervical cancer cells, including the elongated cell shape, increased scattering, down-regulated epithelial markers (E-cadherin and-catenin), and up-regulated mesenchymal marker (vimention). And the complex of E-cadherin and -catenin is adherent junction, one of cell-cell junctions, which will bind with cytoskeletons and enhance the stiffness of cell. Therefore, wild type SiHa cells are expected to be stiffer than KCC3 SiHa cells in cell-cell junction region.
To prove such a hypothesis, we choose the atomic force microscopy, AFM, as study tools with the advantage of observing the cells in their physiological environment, and use the thin film theory, which can solve the problem of substrate effect, to calculate the stiffness of cell junction region. First, we prove the model of thin film theory can be use in the softer materials, PDMS. Then we culture the SiHa cell on the softer materials, PAA Gel, and measure the stiffness of cell junction region. As a result, we found that the thickness of wild type SiHa cells is 3.1 m and thicker than the thickness of KCC3 SiHa cells which is 1.4 m in adjacent region. It suggests that wild type SiHa cells have the complete cell junction, but EMT cells don’t. Moreover, the stiffness of wild type SiHa cells is 1.5 kPa in the adjacent region and stiffer than the other regions which is 0.6-1.3 kPa. However, the stiffness of KCC3 SiHa cells is about 1.0 kPa and similar in all regions. It proves that wild type cells will enhance the cytoskeleton of cell junction region after cell junction forms. According to the two powerful evidences from AFM and thin film theory, we prove that wild type cells form more obvious and stronger cell-cell junction in their adjacent region than EMT cells (KCC3-overexpression SiHa cells).

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