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標題: | 利用雙光子螢光顯微術觀察分子在角質層上滲透情形 Observation of molecular permeation through the stratum corneum by multiphoton fluorescence microscopy |
作者: | Chiu-Sheng Yang 楊秋晟 |
指導教授: | 董成淵 |
關鍵字: | SRB,RBHE,雙光子螢光顯微術,擴散係數,角質層,親水性分子,恐水性分子, diffusion,coefficients,stratum corenum,SRB,RBHE,two-photon excitation fluorescence microscopy,hydrophobic molecule,hydrophilic molecule, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 常見的藥物治療方式有三種,分別是口服、注射以及經皮傳輸治療,由於口服及注射有一些缺點在,而經皮傳輸治療有其特有的優點,主要就是藥物藉由皮膚的滲透,然後使藥物進入到人體的微血管並再達到人體循環系統來達到治療的目的,而我的研究主要就是利用雙光子顯微術來研究觀察藥物在皮膚表皮上滲透,並算出擴散係數,來理解分子在皮膚表面滲透的情形。
我們所使用的螢光分子主要是SRB (Sulforhodamine B) 及RBHE (Rhodamine B Hexyl Ester),SRB是一種親水性分子而RBHE是恐水性分子,這兩種分子在結構及光譜方面非常相似,所以我們選擇這兩種分子來做比較。並用雙光子顯微鏡觀察滲透的動態過程。我們將這兩種分子分別與酒精及磷酸鹽緩衝溶液做混合,此外我們將實驗分為控制組及額外添加3% v/v油酸的實驗組,因為油酸本身有改善皮膚滲透的能力,所以我們要比較含油酸跟沒含油酸的實驗結果差異。而我們發現不論是隨著SRB、RBHE滲透路徑主要都是藉由細胞與細胞的間隙,但是在細胞內RBHE的滲透能力優於SRB,而油酸的加入,增強了分子滲透的能力,但是同時也增加了皮膚的異質性,使得角質細胞形狀明顯的改變。 此外,現今有很多近似角質層結構的模型,理想均質膜的模型就是其中一種,我們將角質層假設成一個均質膜,藉由Fick’s Law和連續方程,得到一個擴散方程,再藉由邊界條件可以得到一個濃度與時間和位置有關的擴散方程解,方程式裡面有個係數叫擴散係數,而我們想要得到SRB、RBHE在皮膚角質上擴散係數的值,我們用Matlab跟Labview去做結合,針對影像每個像素(pixel)去做擬合,我們在同個深度7個時間點擬合出擴散係數,而我們得到了擴散係數分布圖,並得知無論是SRB還是RBHE,細胞與細胞間隙的擴散係數是大於細胞內的,並針對單一角質細胞去看擴散係數在細胞間隙與細胞內隨著深度的差異,而結果顯示不論是細胞內還是細胞間隙,SRB和RBHE的擴散係數都隨著深度逐漸下降。 Currently there are three commonly applied methods for drug delivery including oral delivery, hypodermic injection and transdermal methodologies. Unfortunately, there are some disadvantages associated with oral delivery and hypodermic injection, therefore, in this research we focused on transdermal drug delivery system that has a handful of merits over conventional methods. The drug can diffuse into the deeper subcutaneous depth in the skin tissue by the process of permeation, reach the blood capillary, and finally enter the circulatory system for the therapeutic treatment. We utilized two-photon fluorescence microscopy to study the drug permeation on stratum corneum (SC) in order to calculate the diffusion coefficient and understand the detailed permeation process in skin specimens. The two fluorescent probes including SRB (Sulforhodamine B) and RBHE (rhodamine B hexyl ester) were served as the modeling of hydrophobic and hydrophilic drug agents, respectively, and were selected based on their similarity in molecular structure, and fluorescence emission range. These two fluorescent probes were used for the purpose of comparison and were applied in the following manner. We first mixed SRB with ethanol as well as with PBS as the control group combination. Secondly, we mixed RBHE, separately, with ethanol, PBS, and 3% v/v oleic acid, and used these combinations as the experimental group. Due to the fact that oleic acid can enhance the ability of drug permeation in skin, we compared the results with and without oleic acid. Our analytic results suggested that both SRB and RBHE reached the deeper subcutaneous layer via intercellular route. In addition we discovered that the ability of permeation of RBHE in intracellular area is better than that of SRB. The addition of oleic acid not only enhances the molecule’s permeative ability but also increase its heterogeneity and the revelation of the shape of the cell. Furthermore, there are several models illustrating the physical structure of stratum corenum. In our theoretical modeling we treated stratum corenum as a homogeneous membrane. By Fick’s law and the continuous equation we can obtain a differential diffusion equation. There is a unique diffusion coefficient associated with the diffusion equation for both the permeation of SRB and RBHE in the stratum corenum of skin. For both the permeation of SRB and RBHE we combined the Matlab with Labview, analyzed every pixel of images, fitted the diffusion equation, and obtained the diffusion coefficient from the same depth at seven different time points. Finally, we obtained the distribution diagram and recognized that the value of diffusion coefficient in intercellular domain is higher than that in the intracellular region for both SRB and RBHE. The difference of diffusion coefficients between intracellular and intercellular area were observed at different depths for particular type of cells in stratum corenum, and the results showed that the diffusion coefficients for SRB and RBHE decrease with the increasing depth both intracellular and intercellular regions. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61347 |
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顯示於系所單位: | 應用物理研究所 |
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