以原子力顯微鏡量測晶片上蛋白質間之作用力及癌症藥物對細胞楊氏係數之影響

Abstract

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.