利用飛秒雷射脈衝選擇性誘導細胞死亡及其脫離微製造細胞培養基板之研究

Abstract

仿生裝置的研發已成為細胞行為研究上不可或缺的技術之一，我們的研究目的是對於在細胞培養基板上建構一個活體細胞網絡所提供一種新的研究方法，諸如此類的仿生裝置，有助於控制細胞功能，例如；發育、増殖、遷移、分化及死亡並且扮演非常重要的角色。為了實現細胞基底裝置，在不影響周圍細胞的情況之下，控制誘導特定的細胞死亡是非常重要的。因為細胞的生長是一個非常複雜的過程，其中包含了許多如上述之細胞功能，因此細胞的狀態總是隨著時間改變而不同；在此當細胞完成了他們的任務，他們會進行凋亡並且被其他細胞取而代之，而這種現象也經常在細胞的生長循環當中自然發生。 本項研究之目的為利用飛秒雷射燒融，找到在特定的條件情況下能引發特定單一目標細胞的死亡並移除，進而找尋不同雷射參數與HepG2細胞死亡及其脫離之間的關係。微型處理後的細胞培養基板可有效控制細胞於特定的幾何圖 v 形內並有助於目標細胞的持續監測及追蹤。而這些細胞可型成細胞單層並被有效的形成線型排列。 當高強度的飛秒脈衝雷射被牢牢的聚焦在單一活體細胞內，經常伴隨多光子吸收。如果雷射能量足夠高進而產生空化氣泡使細胞體被撐破，進而導致細胞內知有毒物質釋放到外在環境。此情況下，與之周圍細胞將接收致命的損傷，反之，在相對低的雷射能量情況下，可在非常短的時間內誘導單一目標細胞的死亡並且無影響周遭細胞的持續生長。我們發現有百分之九十六之目標HepG2細胞將會脫離微處理圖案。 我們的方法應用飛秒雷射對於控制細胞的死亡及其脫離原特定位置將會對於未來仿生基板的發展上有重大的貢獻，並且在不久的將來對於重建移除特定的細胞或者細胞死亡的研究上扮演重要的角色，例如；細胞的在生及死亡。此外，在時空上控制細胞及組織的損傷並移除特定的細胞也將會在細胞的再生研究上受到高度的重視。

Construction of bio-functional devices has become one of the most important technologies for studying cellular behaviour. We aim at providing a new methodology to construct living-cell network on a culture substrate toward cell-based devices. Such devices may play an important role in controlling cellular functions such as development, proliferation, migration, differentiation and death. In order to realize the cell-based devices, induction of cell death without affecting the neighbours should be controlled. Because cell maturation is a sophisticated process involving many cell functions, the condition of individual cells is varying during this period. Furthermore, when the cells finishing their roles, they kill themselves to make places to other kinds of cells for maturing tissues. The types of cell death frequently occurs naturally in normal tissue maturation cycle. The purpose of this thesis is to find out a condition for inducing death of individual cells to remove them by utilising femtosecond laser ablation. The relation among laser parameters, cell death and the detachment of dead cell was investigated with HepG2 cell iii (human liver hepatocellular carcinoma cell line). The micro-fabricated cell culture substrate was applied to monitoring individual cells. The cells can be arranged linearly as a monolayer on a micro-pattern. When intense femtosecond laser pulses are tightly focused into a single living cell, ablation is induced as a result of multi-photon absorption. If the laser power is enough high to generate cavitation bubbles, bursting of a single cell is induced to release intracellular toxins to extracellular environment. In this case, surrounding cells receive fatal damages. In contrast, a relatively low power was set just to kill a target single cell in short period without affecting adjacent cells. We found that the target HepG2 cells were detached from the micro-fabricated pattern with 96% reproducibility. Our methodology for control of specific cell death and their detachment by utilising femtosecond laser will contribute to the development of cell-based devices. It will play a significant role in study of selectively controlling cell functions, for example, cell renewal and cell death in near future. In addition, this ability for temporally and spatially controlling cellular and tissue damages and removing an individual specific cells will receive much attention from the view point of regeneration studies.