以微流體晶片研究人類肺癌細胞之趨電性行為

Abstract

趨電性，指貼覆的活體細胞，對具有生理強度之直流電場反應產生的趨向性爬行。在本研究中，我們發展不同的微流體細胞培養晶片，並搭配顯微系統，將其應用於人類肺癌細胞的趨電性觀測上。我們設計並製造了一種可以提供三種不同電場強度的單通道微流體晶片，並研究肺癌細胞對不同強度的電場的趨電性反應。我們以軟體模擬電場在晶片的細胞培養微腔體中分佈的情形，並與實際量測的數據比對，可觀察到在微腔體中的電場呈現均勻分佈。用於此研究的人類肺腺癌細胞株有兩種，分別是具有低轉移能力的CL1-0細胞株，及其具有高轉移能力的CL1-5子細胞株。兩細胞株在外加電場74-375mV/mm下展現截然不同的反應。CL1-5細胞朝向正極爬行，且細胞本體逐漸垂直於電場方向排列。反之，CL1-0沒有表現明顯的趨電性行為。 為了瞭解生理直流電場對細胞基因表現的全面性影響，在本研究中我們採用了微陣列分析法。此微陣列分析使用Affymetrix公司的GeneChip。為了收取分析所需的大量細胞樣本，我們設計並製造了一種大尺寸電場晶片(LEFC)。用於分析的細胞施加以0(對照組)或300mV/mm(實驗組)強度的電場兩小時。將電場刺激下顯現表現差異的基因比對KEGG及BioCarta資料庫，以探討電場可能影響之訊息傳遞路徑。對CL1-5樣本的分析結果顯示，受電場調控的基因與adherens junction、 telomerase RNA component gene regulation及tight junction有高度相關性。對CL1-0樣本的分析結果則顯示，受電場調控的基因與translation、purine metabolism及 cell apoptosis有關。換言之，CL1-0的受電場調控基因與細胞貼覆及細胞能動性並沒有顯著的相關性。此外，在CL1-5的顯著表現差異基因中，有相對高比例的基因產物為膜蛋白，但在CL1-0的顯著表現差異基因中沒有觀察到膜蛋白基因。此結果顯示膜蛋白可能與細胞的趨電性行為有相關性。 我們的研究證實具高轉移與低轉移特性的肺癌細胞，不僅在趨電性的行為表現程度上有顯著差異，其受電場調控的基因表現變化也不同。某些受電場調控基因已知與趨電性有關，其他則是首次被觀測到。此研究結果顯示，趨電性與癌細胞的轉移性具有正相關，且電場作用的機制牽涉到其對基因表現的調控。因此，藉由建立受電場調控基因之蛋白質產物間交互作用的網路，並考慮CL1-0與CL1-5細胞原有的膜蛋白表現差異，在本研究中我們進一步預測了可能具有感測電場能力並開啟訊息傳遞的膜蛋白。

Electrotaxis is the movement of adherent living cells in response to a direct current (dc) electric field (EF) of physiological strength. In this work, microfluidic cell culture chips were developed to be used for long-term electrotaxis study on a microscope. The cellular response under three different electric field strengths was studied in a single channel microfluidic chip. EF inside the micro-chamber was numerically simulated and compared to the measured value. Lung cancer cell lines with high and weak metastasis potential, CL1-5 and CL1-0, respectively, were used to demonstrate the function of the multi-field chip (MFC). The two cell lines exhibited greatly different response under the applied EF of E = 74-375mV/mm. CL1-5 cells migrated toward the anode while CL1-0 cells did not show obvious response. Under the applied EF, cell orientation was observed accompanying the cell migration in CL1-5. To understand the transcriptional response of CL1-5 and CL1-0 cells to a dcEF, microarray analysis was performed in this study. Affymetrix GeneChip was used for microarray analysis. A large electric-field chip (LEFC) was designed, fabricated, and used for sample collection. CL cells were treated with the EF strength of 0mV/mm (the control group) and 300mV/mm (the EF-treated group) for two hours. Signaling pathways involving the genes that expressed differently between the two groups were revealed. In CL1-5, it was shown that the EF-regulated genes highly correlated to adherens junction, telomerase RNA component gene regulation, and tight junction. In CL1-0, the same analysis revealed that the EF-regulated genes were mainly involved in translation, purine metabolism, and cell apoptosis. In other words, the EF-regulated genes in CL1-0 did not show strong correlation to cell adhesion and migration. We further observed a high percentage of significantly regulated genes which encode cell membrane proteins in CL1-5, suggesting that dcEF may directly influence the activity of cell membrane proteins in signal transduction. However, the dcEF did not have significant influence in membrane protein encoding genes in CL1-0. Our study demonstrated that CL1-0 and CL1-5 not only showed distinct activities induced by the dcEF, they also showed different gene expression changes. Some of the EF-regulated genes have been reported to be essential whereas others are novel for electrotaxis. Our result confirms that the regulation of gene expression is involved in the mechanism of electrotactic response. By building an interaction network of EF-regulated genes and considering the original gene expression differences of the two cell lines, we proposed several membrane proteins that may sense the EF signal and cause the different electrotactic responses between the high and low metastatic cancer cells.