利用拉曼光鉗技術觀測單一細胞之氧化傷害過程

Abstract

在生物體內複雜的生化反應中，可能產生具有高度化學反應活性的自由基 (free radical)。而當體內的抗氧化機制與自由基的產生速率失去平衡時，過多的自由基可能對細胞的脂質、蛋白質與DNA造成所謂氧化傷害 (oxidative injury)，導致各種相關疾病的產生。探討自由基攻擊細胞的過程，將有助於了解和自由基相關疾病的的生化機制甚至幫助其預防治療。然而過去自由基攻擊細胞造成的生化變化從未在「單一細胞」的層次進行研究，也從未能透過振動光譜技術在「分子層次」做「即時」的觀測。在本研究中，我們利用自行架設的「共焦拉曼光鉗系統」(confocal Raman tweezers)，在溶液中抓住單一活酵母菌，並動態即時地觀測單一活細胞在自由基存在下之拉曼光譜變化。另外，再以POPC所製備的微脂粒 (liposome) 進行相同實驗，也探討抗氧化劑-維生素C之作用。實驗中發現，在自由基存在下，代表碳鏈雙鍵 C=C 的1651 cm-1 peak與 =CH 的1266 cm-1 peak之強度出現明顯的連續下降趨勢，同時其半高寬亦會產生顯著的增大現象。但是代表碳鏈單鍵 -CH2 的1441 cm-1 peak與1300 cm-1 peak 並無明顯變化。以不飽和脂肪酸POPC所製備的微脂粒在相同實驗下亦觀察到類似的變化。整個實驗結果可以用自由基引起脂質過氧化 (lipid peroxidation) 加以解釋。我們也在光譜上觀測到抗氧化劑-維生素C對自由基反應的抑制，更進一步支持以上的解釋。

For the first time, the oxidative stress response of single cells and the radical scavenging effect by anti-oxidants were observed and monitored in real time. A tightly focused laser beam was employed to capture and trap individual yeasts and, in the mean time, to generate Raman emission of the trapped object. The kinetic series of Raman spectra were recorded to follow the real-time chemical change of the trapped object under externally applied stresses. Four distinct bands (1266, 1300, 1441, 1651 cm-1) are identified in the Raman spectrum of single yeasts and are assigned to =CH bending, CH2 twisting, CH2 bending, and C=C stretching, respectively. Under an oxidative stress produced by the Fenton’s reaction, the intensities of the Raman bands at 1266 and 1651 cm-1 were found to decrease along with time while the other two remain the same. The diminish of the Raman bands associated with C=C double bonds (1266 and 1651 cm-1) was explained by the per-oxidation damage of unsaturated lipid on cell membranes induced by hydroxyl radicals. Same trends observed on liposomes made of unsaturated lipid (POPC) further supports our explanation. Remarkably, the slower decrease of the Raman bands with the addition of vitamin C is also consistent with the well established radical scavenging effect of vitamin C. In conclusion, we have observed molecular-level change in real-time on single optically trapped cells under oxidative stress by hydroxyl radicals and their scavenge by anti-oxidants. Our experiment resembles a single-cell level ischemia-reperfusion and signifies a critical advancement on the molecular-level understanding of a clinically important process.