臺灣博碩士論文加值系統

本研究是利用原子力顯微鏡(Atomic Force Microscopy, AFM)，探討固定在晶片上之蛋白質(imm-protein)，與固定在AFM探針上之蛋白質(afm-protein)之間的作用力。此方法得到直接且精準的單一蛋白質間作用力的量測結果。本研究量測T細胞表面接受器(receptor)CD28與B細胞表面接受器CD80間的作用力，以及免疫抑制劑-洋薊酸(cynarin)阻斷兩者結合的結果。經過cynarin阻斷的結果，CD28/CD80間的平均分離力由61.4 pN降至38.9 pN，降低37%。此一量測法和以往用表面電漿共振儀所量測得到的9%的降低率比較，有更大的定量差異。因此，本研究可以利用AFM作為藥物篩選之新方法。
另一方面，本研究也提出一種人體可接受，且較無副作用之長度為33個胺基酸的抗癌胜肽(CB1a)，其在培養皿中已發現對肺癌細胞有明顯的毒殺作用。在此篇報告中，本研究以AFM分析此抗癌胜肽對單一癌細胞的毒殺過程。包括掃描細胞表面形貌，並且將AFM探針壓入細胞，量測細胞膜的楊氏係數，比較加入藥物前後之癌細胞的表面變化情況。正常細胞MRC-5加入25 M CB1a後，楊氏係數沒有太大改變，而癌細胞NCI-H460細胞膜的楊氏係數則在10分鐘內下降至原本的50%以下，由此可以說明此抗癌胜肽作用於癌細胞的選擇性。本研究也比較市面上抗癌藥物-速溶艾黴素(Doxorubicin, Dox)對癌細胞之影響，並進一步比較兩種藥物之毒殺機制的不同。結果發現，CB1a屬於從外部破壞細胞膜，而Dox由內部之生化反應讓細胞凋亡，兩者在表面形貌與細胞物理性質之表現有明顯的不同。

In this dissertation, we demonstrated that the direct and accurate measurement of protein-protein interaction force can be done by atomic force microscopy (AFM) via immobilization of one protein on chip (imm-protein) and another on probe tip of AFM (afm-protein). Preliminarily, we observed the binding between T-cell receptor, CD28 (imm-CD28), and B-cell receptor, CD80 (afm-CD80), with an immuno-suppressive blocker, cynarin. Average unbinding forces were reduced from 61.4 pN to 38.9 pN with a blocking effect of ~37% as compared with ~9% by SPR. Using AFM as a detection tool, the significant quantity differences caused by drug target binding therefore can be applied as an efficient and accurate drug screening or protein-protein interaction measurement.
The other subject in this dissertation is to discuss the drug effect on cell’s mechanical property. A less side-effect and non-harmful custom anti-cancer peptide (CB1a) composed by 33 amino acids was created. The results showed that the peptide drug has cytotoxicity effect on cancer cells in solution (in vitro). The elastic property differences of single cell surface while interacted with anticancer drug CB1a were estimated by AFM. The changes of elastic force of cell with/without CB1a was illustrated and reflected as Young’s modulus. After adding CB1a, the normal cell, MRC-5, almost remained its original Young’s modulus value. For cancer cell NCI-H460, the Young’s modulus reduced to only below 50%. It means that the quantity variation can be considered as a good selectivity of CB1a and provided another cytotoxicity mechanism effect on cancer cell surface. Furthermore, we also compared with the commercial anti-cancer drug, Doxorubicin (Dox), to study the differences of morphology and physical property changes under drugs treating. The results prove that CB1a prefers to damage cell membrane from outside, and Doxorubicin leads cell to death by stopping the functions inside the cells.

中文摘要..………………………………………………………………………i
英文摘要…...……………………………………………………………………ii
誌謝……………………………………………………………………………iii
表目錄…………………………………………………………………………viii
圖目錄..…………………………………………………………….…………ix
一、緒論..………………………………………………………………………1
1.1 研究背景..……………………………………………………………...1
1.2 研究動機與目的..……………………………………………………...3
二、文獻回顧..…………………………………………………………………9
2.1 生物感測器的種類……………………………………………………9
2.1.1 螢光感測………………………………………………………9
2.1.2 酵素連結免疫吸附法…………………………………………10
2.1.3 表面電漿共振…………………………………………………11
2.1.4 石英晶體微天秤………………………………………………12
2.1.5 奈米線…………………………………………………………13
2.2 自組裝單層膜在生物晶片之應用..…………………………………13
2.3 原子力顯微鏡在生物醫學領域之應用.………….…………………15

三、原子力顯微鏡..…………………………………….………………………20
3.1 原子力顯微鏡概論..…………………………………………………20
3.2 原子力顯微鏡之探針..………………………………………………22
3.3 四象限光偵測器..………………………….…………………………23
3.4 生物用原子力顯微鏡..………………………………………………24
3.5 探針彈簧係數之校正..………………………………………………25
四、實驗方法與流程..…………………………………………………………28
4.1 實驗材料..……………………………………………………………28
4.2 實驗設備..……………………………………………………………28
4.3 實驗步驟..……………………………………………………………29
4.3.1 CD28/CD80間作用力與抗過敏候選藥物cynarin的影響…….29
4.3.1.1 晶片清洗..………………………………………………29
4.3.1.2 自組裝單層膜之形成..…………………………………29
4.3.1.3 交連分子膜之形成..……………………………………30
4.3.1.4 蛋白質單層膜之形成..…………………………………31
4.3.1.5力譜量測與力量加載率……………….…………………31
4.3.1.6 蛋白質間作用力的量測..………………………………35
4.3.1.7 加入抗過敏候選藥物cynarin後的分子間作用力……37
4.3.2 抗癌胜肽CB1a對於細胞的影響..……………………………37
4.3.2.1 細胞培養與藥物之細胞毒性..…………………………37
4.3.2.2細胞腫瘤培養……………….……………………………38
4.3.2.3細胞影像掃描……………….……………………………38
4.3.2.4 原子力顯微鏡量測細胞機械性質.….…………………39
五、結果與討論………………………………………………………………51
5.1 cynarin對CD28/CD80結合之影響……………….…………………51
5.1.1 CD28修飾於晶片之表面粗糙度分析…………………………51
5.1.2 CD80修飾於AFM探針之分析………………………………52
5.1.3探討cynarin對CD28/CD80分離力之影響…………………….52
5.1.4 imm-CD28/afm-CD80接觸時間與結合機率之研究…………54
5.1.5 結果討論………………………………………………………54
5.1.6 結論……………………………………………………………59
5.2 CB1a對於正常細胞與癌細胞之影響………………………………60
5.2.1 CB1a對癌細胞與癌細胞腫瘤之抑制能力……………………60
5.2.2 以AFM研究CB1a對正常細胞與癌細胞之外觀變化………61
5.2.3 以AFM研究CB1a對正常細胞與癌細胞之楊氏係數之影響..62
5.2.4 結果討論………………………………………………………64
5.2.5 結論……………………………………………………………69


六、結論與未來展望………………....………………………………………95
6.1 結論…………………………………………………………………95
6.1.1原子力顯微鏡對藥物篩選之研究貢獻………………………95
6.1.2原子力顯微鏡對抗癌藥物之研究貢獻………………………96
6.2 未來展望……………………………………………………………98
七、參考文獻 ………………………………………………………………100

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