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標題: | 電場及壓力等物理性刺激對肺癌細胞表現型的影響 The Effects of Electrical and Pressure Stimulations on the Phenotype of The Lung Cancer Cells |
作者: | Yu-Chiu Kao 高于媝 |
指導教授: | 郭柏齡(Po-Ling Kuo) |
關鍵字: | 趨化性,趨電性,微流道細胞培養裝置,靜水壓,小窩蛋白,水通道蛋白,?胞外調節蛋白激?, Chemotaxis,Electrotaxis,Microfluidic cell culture device,Hydrostatic pressure,Caveolin,Aquaporins,ERK1/2, |
出版年 : | 2016 |
學位: | 博士 |
摘要: | 腫瘤微環境中表現的各種機械特性對癌細胞的轉移有很大的影響,轉移更是導致惡性腫瘤造成死亡的主要原因。例如、在腫瘤周圍的組織中,會存在各種細胞趨化因子的濃度梯度以及組織內直流電場(dcEF),這兩種不同的刺激可能同時出現在相同或相反的方向;因此,癌細胞會受到不同化學濃度梯度以及不同電場方向多種組合的影響。為了研究在相同和相反方向上排列的化學濃度梯度和dcEF如何影響人類肺癌細胞CL1-5的運動特性,我們建構了允許癌細胞暴露於dcEF和上皮生長因子(EGF)濃度梯度的微流道細胞培養晶片。我們發現CL1-5細胞受到直流電場的影響傾往正極方向移動。CL1-5細胞也受化學濃度梯度的影響向EGF濃度高的方向遷移。當CL1-5細胞受到直流電場和相反方向的EGF濃度梯度(0.5μM/ mm)的影響時,隨著電場增加至360mV / mm,CL1-5細胞的移動沒有固定的方向。當電場增加到大於540mV / mm時,電場的影響大於EGF濃度梯度的影響使得CL1-5傾向朝向直流電場正極的方向遷移。在這個微流道細胞培養晶片中我們也證明了兩種趨電性相反的肺癌細胞(CL1-5、A549)皆可以利用直流電場調控細胞的趨化性。
另一方面,組織間質液壓力(interstitial fluid pressure),不管在生理或是病理的過程中,都是很重要的生物指標。較高的組織間質液壓力是大部分腫瘤共同的特徵,但較高的壓力對癌細胞表現型造成的影響仍不清楚。在本篇論文中,我們發展出一套細胞培養系統,能對活體細胞模擬靜水壓力 0 到 20 毫米汞柱,去觀測細胞的行為表現,我們發現升高的靜水壓會導致肺癌細胞CL1-5和A549的移動速度、侵襲能力、細胞體積、絲狀偽足數目增加;從分子生物學的觀點來探討,升高的靜水壓導致細胞水通道蛋白(aquaporin-1)、 蝸牛蛋白(snail)、 黏著斑蛋白(vinculin)、 磷酸化的小窩蛋白(caveolin-1)、 磷酸化的细胞外調節蛋白激酶(p-Erk1/2)表現量增加。由先前的研究得知細胞的移動速度以及體積的增加與aquaporin表現量有關,所以當我們使用小干擾RNA(siRNA)轉染來抑制aquaporin-1時,原本受到靜水壓誘導使得移動速度增加的情況被抑制了;接著使用MEK蛋白激酶抑製劑(PD98059)抑制ERK1/2磷酸化,結果也抑制了水壓誘導aquaporin-1表現量上升以及移動速度上升的情形。最後透過siRNA轉染來抑制caveolin-1,因靜水壓誘導p-ERK1/2使得aquaporin-1表現量上升和移動速度增加的情形也都被抑制了。最後的結論為較高的組織間質液壓力主要是透過 caveolin-1的磷酸化誘導蛋白激酶B(Akt1/2k)的磷酸化進而活化Erk1/2,促使改變面積、絲狀偽足的數目和單隻細胞體積大小,因此可大幅增強肺癌細胞的侵襲力和移動能力。相比之下,正常支氣管上皮細胞在較高組織間質液壓力的環境下對上述的各種影響沒有明顯差異。我們的研究結果發現了一種新的分子路徑,證明了高組織間質液壓力與癌細胞的侵襲能力相關,有助於癌症治療的進步和發展。藉由細胞在體外微環境下運動行為以及表現型的研究,可獲知體內各種微環境條件調控癌細胞轉移的機制,對於癌症的治療,提供新的檢測平台以及臨床參考的知識。 The mechanical characteristics presented in cancer microenvironment have a pivot role in the cancer cell’s metastasis, which accounts for the leading cause of death from malignant tumors. For example, cancer cell migration is known to be driven by the concentration gradients of various cytokines and direct-current electric fields (dcEFs) coexisted in the cell’s microenvironment. To investigate how chemical gradients and the dcEFs that are aligned in the same and opposite directions affect the motility of the human lung cancer cells CL1-5, we built a microfluidic device that allowed the cancer cells to be exposed to both dcEFs and the concentration gradients of epithelial growth factor (EGF). We found that the CL1-5 cells tend to move to the anode when the dcEFs were applied, prefer to migrate against the concentration gradient of the EGF. When both the dcEFs and the EGF gradients were applied to the cells, the chemotactic movement of the CL1-5 cells driven by a EGF concentration gradient of 0.5M/mm was hindered by a contrary-aligned dcEF of 360mV/mm, and was overcame when the electric field was increased to more than 540mV/mm. We also demonstrated electrically modulated chemotaxis of the lung cancer cells A549 using electrotaxis in this device. Besides, most solid tumors are characterized by high interstitial fluid pressure but whether the high pressure affects cancer cell motility remains unclear. In this regard, we found that the elevated hydrostatic pressures(HPs) increased the migration speeds, invasiveness, cell volume, filopodial number and aquaporin-1 (AQP1), Snail and vinculin expression levels, as well as phosphorylation of caveolin-1 and extracellular signal–regulated kinase1/2 (ERK1/2), in the lung cancer cells CL1-5 and A549. The increases of migration velocity and cell volume were associated with the increased AQP1 expression. When we used small interfering RNA (siRNA) transfection to knockdown AQP1, the elevated HP-induced migration acceleration was hindered. Inhibition of ERK1/2 phosphorylation using the mitogen-activated protein kinase kinase inhibitor PD98059 abrogated the elevated HP-induced AQP1 upregulation and migration acceleration in the cancer cells. Caveolin-1 knockdown by siRNA transfection attenuated the HP-induced, ERK1/2-depedent AQP1 upregulation and migration acceleration. Further biochemical studies revealed that the caveolin-1 activation-driven ERK1/2 signaling is mediated by Akt1/2 phosphorylation. By contrast, the elevated HPs had negligible effects on the migration speed and volume of normal bronchial epithelial cells. Our results disclose a novel pathway relating high IFP to the invasiveness of cancer cells and highlight potential targets to hinder cancer cells spreading. Collectively, these novel findings of the motility and phenotype of the lung cancer cells subject to in vitro microenvironments recapitulating the complex conditions in vivo should shed light in the understanding of cancer cell metastasis and provide a new testing platform and knowledge useful for clinical applications. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/60148 |
DOI: | 10.6342/NTU201603770 |
全文授權: | 有償授權 |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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