於正常細胞中轉染致癌基因對APTES基板表面細胞印痕之影響

The Effect of Oncogenes Transfection into the Normal Cells on The Cell Imprints Left on γ-APTES Substrate Surface

陳珮驊(Pei-Hua Chen)

指導教授 ： 吳幼麟

共同指導教授 ： 徐中平(Chung-Ping Hsu)

國立暨南國際大學/科技學院/生物醫學科技研究所/碩士(2010年)

摘要

本篇論文中, 我們將正常細胞以及經過致癌基因轉染之正常細胞培養至經由 γ-APTES (3-aminopropyltriethoxysilane)對表面做化學處理後之二氧化矽晶片表面上，然後透過原子力顯微鏡觀察，細胞後於奈米尺度下觀察之表面形貌。由於細胞力學之因素，細胞會將力量施加於γ-APTES之表面上，而癌症細胞與正常細胞之細胞力學必定不同。故經由本論文提出之方法亦可將其細胞與基板之交互作用現象利用原子力顯微鏡完整呈現。本實驗室先前曾以前述方式針對培養於γ-APTES表面之正常和癌細胞直接偵測，發現癌症細胞之印痕深度相較於正常細胞深度來的深。而過去文獻中的報告指出，致癌基因會對基板表面造成較大的細胞抓附力。因此，本論文嘗試將正常細胞中之致癌基因大量表現，使正常細胞轉型後利用原子力顯微鏡觀察其施加於γ-APTES表面上印痕深度是否變深。
本論文中我們選用了K-Ras，Src，YES，Myc，Ets2，Met六種致癌基因，以及控制組的pcDNA3.1。利用原子力顯微鏡觀察送入致癌基因之正常細胞與沒有經由致癌基因處理之細胞以及與控制組細胞比較。經由細胞移除過程後，我們發現當細胞大量表現致癌基因時其細胞印痕改變成只有在細胞周圍產生，而控制組之印痕與沒有做任何處理之正常細胞印痕相同。此結果與我們的預期相同，因此我們推測細胞周圍產生之環狀印痕變化應該是由於致癌基因之大量表現所造成。
細胞抓附力為目前最熱門研究之一，目前針對於細胞抓附力之研究，皆以模擬或是預測之方式進行分析，由於受限於製程方法，這些方法只單方面直接對細胞周圍的抓附現象進行探討，並無法觀察細胞整體之情形。本實驗所提出之量測方式則可以觀察細胞整體抓附力之型態，此為先前之研究所無法觀測到的。本論文之實驗結果或許可以幫助研究人員對於癌症細胞之侵犯與轉移能力之機制有更進一步地瞭解。

關鍵字

γ-APTES ； 致癌基因 ； 細胞抓附力

並列摘要

In this thesis, we investigated the post-cell-removal surface morphology profiles for the normal cells as well as nomal cells treated with oncogenes transfection that were cultured onγ-APTES modified silicon dioxide surface. By taking advantage fo its nanoscale measurement ability, we used atomic force microscopy to obtain the post-cell-removal surface morphology profiles. Since force would be exerted by cells to theγ-APTES substrate surface beneath according to the cell dynamics, it is certain that the surface morphology profiles would be different for normal cell and cancer cell due to the nature of the two cells are different. According to the previous experimental results obtained in our lab., the cancer cells exhibited a higher traction force at the circumference than did the normal cells. A lot of papers in the literature reported that the high cell traction force in cancer cells was related to the transfection of oncogenes. Therefore, in this work we used atomic force microscopy to measure the post-cell-removal surface morphology profiles for normal cells and normal cells after oncogenes transfection..

The oncogenes K-Ras，Src，YES，Myc，Ets2，Met were chose for normal cells transfection in this work, and the pcDNA3.1 was used as the control. Comparing the post-cell-removal surface morphology profiles of the normal cells before and after oncogenes transfection, deeper trench was found along the circumference of the cell imprints when oncogene overexpression in the normal cells occurred. On the other hand, the trasfection of pcDNA3.1 into the normal cells did not change the cell imprint. These results confirm that the deeper trench along the circumference of the cell imprints is mainly caused by the oncogenes transfection.

In recent years, the cell traction force has become a very important research topic and many methodologies have been developed for the related investigation. However, most methodologies dealt with only those traction force around the cell periphery but not the whole cell and some investigations were carried out based on simulation or prediction. Therefore, there are still many challenges remained.. In this work, with the help of atomic force microscopy we could clearly observe the post-cell-removal surface morphology profiles, from which the cell adhesion properties at both the periphery as well as central region can be discerned. This technique can help us understand more clearly about the relationship between oncogenes and cell metastasis as well as invasion.

並列關鍵字

γ-APTES ； oncogene ； cell traction force

參考文獻

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