| Abstract: | 細胞進行下沉代謝或藥物代謝時常可伴隨活性氧分子（reactive oxygen species）的產生，這些活性氧分子如無法有效清除，將形成氧化壓力（oxdative stress），造成細胞傷害，進而死亡。在細胞受到氧化傷害的過程中，常可觀察到細胞表面發生特殊的形態變化，即所謂發泡現象（membrane blebbing）。本實驗主要目的在探討氧化傷害與小泡生成的關係，尤其著重在細胞架構蛋白-肌動蛋白（actin）與管蛋白（tubulin）兩者在小泡生成時扮演的角色。除此之外，胞內重要抗氧化分子：維生素C，保護此種形態變化的作用也是本實驗目的之一。本實驗共分兩部份：
第一部分：探討肝細胞膜小泡生成過程中，細胞膜結合架構蛋白（membrane-associated cytoskeletal proteins）扮演的角色。肝細胞在48小時培養後，分別加入2.0mM t-buty1 hydroperoxide (t-BHP)或2.0 mM diamide（硫醇氧化劑）誘發細胞膜小泡生成，並分別於0、15、30、60分鐘取樣，分析細胞膜硫醇流失程度；乳酸去氫□滲漏；□胱甘□濃度變化；脂質過氧化；肌動蛋白與管蛋白變化；並利用雷射共軛焦螢光顯微鏡觀察肝細胞中肌動蛋白及管蛋白的變化。結果顯示t-BHP或diamide均可造成細胞膜蛋白硫醇大量流失與胞內GSH快速氧化，亦顯著導致乳酸去氫□滲漏，但t-BHP誘發的滲漏程度大於diamide；在脂質過氧化方面，則僅t-BHP處理組產生明顯的脂質氧化情形；至於肌動蛋白與管蛋白單體均可見減少且呈現時間依賴效應；在雷射共軛焦螢光顯微鏡觀察中，亦顯現這兩種架構蛋白與細胞膜起泡有關，但兩者在小泡生成的過程中扮演的角色並不完成相同，可能管蛋白較肌動蛋白重要；如果細胞在t-BHP或diamide加入前預先以dithiothreitol（DTT，硫醇還原劑）處理，則diamide 所造成的傷害顯著的受到抑制，但DTT對t-BHP所誘發的傷害則僅有部分保護效果。本實驗顯示細胞形態的改變確與胞內硫醇，尤其是細胞架構蛋白硫醇有關。
第二部份：探討不同濃度維生素C 在2.0mM t-BHP處理下，是否可以保護細胞的形態。結果顯示：500μM維生素C預先處理下，細胞形態受損與細胞膜蛋白質硫醇的情況有明顯的改善；但在脂質過氧化方面則無明顯抑制作用，顯示維生素C可以減少t-BHP所誘發的小泡，而且其機制可能與維生素C保護膜上蛋白質，尤其是細胞架構蛋白免於氧化傷害有關。
綜合以上兩個實驗結果，我們認為：（一）氧化緊迫下，細胞膜蛋白質硫醇的氧化在肝細胞發泡過程中可能扮演著重要的角色；（二）肌動蛋白及管蛋白與細胞形態的維持有密切相關，其中管蛋白扮演的角色可能比肌動蛋白來的重要；（三）維生素C可抑制細胞在氧化緊迫下發生形態改變。
Normal metabolismn and drug metabolism in cellular microenvirement can lead to the production of reactive oxygen species. Once these reactive these reactive oxygen species cannot be eliminated, cells will be damaged and leads to membrane morphological changes, such as the bleb formation. The purpose of this study was to examine the cellular surface blebs of oxidative damage. The important is cytoskeletal proteins-actin and tubulin acts in cellular surface blebs. Besides, intracellular important antioxidants: ascorbic acid, the study was compare the protective effects of ascorbic acid on the membrane morphological changes. The study was two section.
In the first section of the study, the examine the role of membraneassociated cytoskeletal proteins during the process of hepatocytes blebbing. After developing 48 hours, hepatocytes enters 2.0 mM t-butyl hydroperxide (t-BHP) or 2.0 mM diamide (thiol oxidant) to induced cell blebbing. The samples collected at 0, 15, 30, 60 minutes to analyze cell about membrane protein thiol deletion; the leakage of lactate dehydrodenase; the change of glutathione; the lipid peroxidation; the variation of actin and tubulin, and use Confocal fluorescent microscope to observe the change of actin and tubulin in rat hepatocytes. The result indicates the t-BHP or diamide both cause the mass loss about the membrane protein thiol in hepatocytes, the fast oxidization about glutathione in hepatocytes, the leakage of lactate dehydrodenase, but the level of t-BHP is larger than of diamide. In the aspect of lipid peroxidation, only the t-BHP produces apparent change. As to actin and tubulin, they both find fall-off and present time-dependency effects. The result in Confocal fluorescent microscope also show these two cytoskeletal proteins are related to cell blebbing, but they don''t play the same role during the porcess. Tubulin may be more important than actin. If the study predispose dithiothreitol (DTT, thiol reductant) before adding t-BHP or diamide, then the damage by the diamide is repressed, but just partly protection for the damage by t-BHP. The study reveals the change of cell type is related to thiol in cell, especially cytoskeletal proteins.
In the second part, the explore ascorbic acid if protecting cell type under disposing 2.0 mM t-BHP. The result finds that the damage of cell type and the condition of membrane protein thiol improve evidently under predisposing 500 μM ascorbic acid. But the aspect of lipid peroxidation is not repressed, it shows ascorbic acid can decrese the blebbing derived by t-BHP, and the mechanism is related to ascorbic acid which protects membrane protein, especially cytoskeletal proteins, from oxidization.
In a word, we think: 1)Under oxidization stress, the oxidization of membrane protein thiol may play more inportant role in cell blebbing. 2)Actin and tubulinare related to the maintenance of cell type, and tubulin may be more important than actin. 3)ascorbic acid can repress cell type to change under oxidization stress. |
