- 學位論文
高量吞噬磷脂質DOPE對細胞活性之影響
Influence of cellular activity by high-level treatment of phospholipid DOPE-loaded liposomes
摘要
奈米脂微粒在生醫產業上的應用很廣泛，依照需求的不同而可以在奈米脂微粒上進一步地修飾，而產生不同的功能。其中以正價脂質為主體的正價奈米脂微粒，利用帶電性與質體DNA或是siRNA結合，而形成的脂質體-DNA複合物 (lipoplex)，常作為良好的轉染試劑。除了正價脂質以外，正價奈米脂微粒的組成通常會與不同種類的中性脂質依不同比例混合，像是膽固醇 (cholesterol) 或者是磷脂質DOPE (dioleylphosphatidylethanolamine)。不管在實驗室或者是產業上，正價奈米脂微粒的崛起，不得不讓科學家們去正視正價奈米脂微粒對於細胞生理上的影響，因此許多文獻開始探討正價奈米脂微粒對細胞造成毒性的研究，然而主要研究方向集中在正價脂質上，對於中性脂質的探討卻不多。 DOPE被稱為輔助者脂質 (helper lipid)，由於其本身錐狀的結構，使得細胞內體膜產生不穩定的情況，進而使含有DOPE的正價奈米脂微粒，所承載的DNA或者是藥物可以順利的進入到細胞內，增加藥物效用或轉染的效率。因此，像是Lipofetamine 2000與Lipofectin這兩種轉染試劑的組成中都含有DOPE。先前研究曾指出含有DOPE的正價奈米脂微粒，對細胞造成較大的毒性，然而DOPE對細胞造成那些影響，以及它作用的詳細機制，目前尚未釐清。 本論文利用正價脂質GEC-chol{3β-[N-(2-guanidinoethyl)carbamoyl]choleste -rol}、DOTAP (1,2-dioleoyl-3-trimethyl-ammonium-propane)，分別與中性脂質cholesterol與DOPE合成兩組正價奈米脂微粒，GCC、GCE與DOC、DOE。利用這四種奈米脂微粒，處理小鼠巨噬細胞RAW264.7與人類子宮頸上皮癌細胞HeLa，探討細胞吞噬的效率、細胞毒性、活性氧自由基產生的量以及細胞膜受損程度，進一步驗證含有DOPE的奈米脂微粒對細胞造成的傷害，確實比膽固醇的組別還高。除此之外，中性脂質的不同，使得GCC與GCE在細胞中的螢光分布有所差異；另外發現細胞長時間吞噬正價奈米脂微粒後，會將過多的膽固醇釋放到胞外，而DOPE卻沒有這樣的情形。 本論文除了進一步探討含有DOPE正價奈米脂微粒的細胞毒性，同時也在中性脂質膽固醇與DOPE之間特性的差異，有了新的發現，並且對於DOPE在細胞毒理以及生理上有更深的了解。
並列摘要
Cationic liposomes have been referred to an effective carrier of DNA, siRNA , antigen or drug in therapeutics as well as biotechnology. They usually compose of cationic lipid and neural lipid, such as cholesterol or dioleylphosphatidylethanolamine (DOPE). Because of its wide use in laboratory and clinical, the toxicity of cationic liposome must be clearly investigated. However, most researches were focused on cytotoxicity of cationic lipid rather than neutral lipid. DOPE is a common helper lipid in liposomes due to its fusogenic property. The structure of DOPE is conical shape which can destabilize the membrane bilayer of plasma membrane or endosome so that the cargo incorporated into liposomes releases efficiently into cytosol. As a result, DOPE can greatly increase the transfection efficiency of cationic liposomes. Therefore, several drugs and transfection reagents, such as lipofectamine 2000 and lipofectin, are formed with DOPE. In previous study, the formulation with different cationic lipids and DOPE resulted in a high toxicity toward macrophages. The toxicity is reduced by replacement of DOPE with dipalmitoylphosphatidylcholine (DPPC). Nevertheless, the mechanism how DOPE involved in cell toxicity is not clear so far. In this research, cholesterol-based cationic lipid GEC-chol and DOTAP were incorporated with DOPE (termed as GCE or DOE) or cholesterol (termed as GCC or DOC) respectively. Different kinds of liposomes shown above were investigated to influence cells such as cellular uptake, viability, reactive oxygen series production and cellular membrane damage using mouse macrophages RAW 264.7 and human cervical epithelial cancer cells HeLa. Results showed that the cytotoxicity of DOPE-loaded cationic liposomes were higher than cholesterol-loaded cationic liposomes. In addition, intracellular fluorescence localization between GCC and GCE were different and extra cholesterol was pumped out from RAW264.7, but DOPE not. In conclusion, the distinguishing characteristics between cholesterol and DOPE in cationic liposomes and the significance of DOPE in toxicology, pharmacology or physiology are revealed.