臺灣博碩士論文加值系統

跨膜運輸是維持正常細胞生理所必須，除了是生物物理的重要議題也和許多生醫應用如：藥物輸送、基因治療等有密切相關。過去研究曾提出脂質膜 (lipid membrane) 在熱擾動下可自發性形成奈米級暫時性孔洞 (spontaneous pore formation)，以解釋一些異常跨膜運輸的現象。但由於其尺度小、存在時間短，研究脂質膜上之暫時性孔洞仍是挑戰。因此過去有團隊提出以電脈衝、表面張力、照射強光等方式在巨型微脂體 (giant unilamellar vesicle) 上產生微米尺度之暫時性孔洞，並增加溶液黏滯度 (viscosity) 延緩孔洞關閉，以達到即時觀測暫時性孔洞的目的。在此，我提出以高靈敏的螢光偵測技術，測量螢光染料分子經由暫時性孔洞流入單一巨型微脂體，造成之微脂體內螢光強度動態變化，來探討巨型微脂體自發形成之暫時性孔洞。即時記錄螢光強度出現的間歇性螢光突波 (fluorescent burst)，螢光突波的共同特徵是有一快速 (<1 s) 的上升，接著是約數秒的緩慢下降段，另外突波高度則有兩種主要分布。此特徵可以歸因於螢光染料分子藉由膜上的暫時性小孔流入微脂體內，及孔洞關閉後持續雷射激發的光漂白 (photobleaching) 現象。螢光分子通過膜上小孔的流量 (flux) 是與小孔的尺度及開啟時間有關，根據 Berg 和 Purcell 的理論分析螢光突波，可以得到 POPC 巨型微脂體上形成的暫時性孔洞開啟時間約 0.27 s，孔洞則有兩種主要尺度 (半徑約 12.8 nm 和 34.4 nm)，而混有 3 mol% 和 10 mol% 膽固醇的微脂體上形成的暫時性孔洞也有兩種相似的主要尺度以及開啟時間。進一步分析發現，相較於純 POPC 的微脂體，混有膽固醇的微脂體上較小的暫時性孔洞 (半徑~10 nm) 有較大的邊緣張力 (edge tension, 1 pN vs. 0.85 pN)。這是第一個由螢光分子洩漏實驗決定脂質膜上暫時性奈米級孔洞的尺度及開啟時間的研究，未來預計可開啟一系列相關研究。

Transmembrane transport is not only an important subject in biophysics but also intimately related to varied practical applications such as drug delivery, gene therapy, et al. While a large body of recent studies have shown that transient pores can form on lipid membranes either spontaneously or subject to stress, our understanding of transient pores remains elusive mainly due to the difficulty to characterize their fundamental properties. Here we report a novel experimental approach to determine the opening time and the dimension of transient pores formed spontaneously on giant unilamellar vesicles (GUV). GUV made of phospholipids (POPC) were immersed in a solution of fluorescent probes (carboxyfluorescein), and a sensitive lock-in amplified confocal detection scheme was employed to measure the fluorescence change at the center of the GUV that arises from the leakage of fluorescent probes through a transient pore. Notably, the temporal trace of the fluorescence intensity comprised numerous intermittent fluorescent bursts, and each of which generally has a rapid rise (~0.2 s) and a slow decay (a few seconds). The rapid rise of individual fluorescent burst was attributed to the influx of fluorescent probes through a transient pore, and the slow decay to the subsequent photo bleaching of fluorescent probes that were enclosed in the GUV, respectively. We then adopted Berg and Purcell’s approach to analyze the temporal profile of these bursts, and found that these transient pores generally have an opening time about 0.27 s and have two prominent dimensions (radius = 12.8 nm and 34.4 nm) respectively. Further studies show that transient pores found on GUV comprising cholesterol (3 mol% and 10 mol%) exhibit comparable characteristics. Nevertheless, the edge tension of small pores on GUV comprising cholesterol seems slightly larger relative to that of small pores on POPC GUV (~1 pN vs. 0.85 pN). Our approach makes possible the determination of the essential characteristics of transient pores formed spontaneously on lipid membranes, and should open a new route to study fundamental aspects of transient pores formed either spontaneously or by external stress.

摘要 i
ABSTRACT ii
致謝 iii
目錄 iv
圖目錄 vi
表目錄 viii
第一章、 背景 1
1-1 細胞膜輸送 (membrane transport) 1
1-2 被動穿膜運輸 (passive transmembrane transport) 5
1-3 暫時性孔洞 (transient pore) 之文獻回顧 10
1-4 暫時性孔洞之能量學 19
1-5 研究動機與架構 22
第二章、 研究方法 23
2-1 藥品 23
2-2 單點共聚焦螢光偵測系統 (single-point confocal fluorescence system) 24
2-3 巨型微脂體製備 26
2-4 單點共聚焦螢光偵測系統的量測 28
第三章、結果與討論 30
3-1 巨型微脂體 30
3-2 單點螢光隨時間變化-尖波 (spikes) 32
3-3 尖波之訊號分析 36
3-3-1 定義尖波之上升高度 36
3-3-2 選取尖波並平均 37
3-3-3 尖波之曲線擬合 37
3-3-4 估算孔洞大小 38
3-4 脂質膜不同膽固醇濃度對孔洞的影響 42
3-5 比較脂質膜不同膽固醇濃度的邊緣張力 44
3-5-1 脂質膜 (POPC) 的邊緣張力 45
3-5-2 不同膽固醇濃度的邊緣張力 46
第四章、結論與未來展望 48
第五章、參考資料 49

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