臺灣博碩士論文加值系統

本研究是一方面利用原子力顯微鏡(AFM)來量測非貧血大鼠和貧血大鼠的紅血球細胞膜剛性係數；另一方面，以微製程技術設計製造三角尖端結構晶片，並施以直流脈衝電壓，依此結構的壓縮造成電場疏密不均，來裂解非貧血大鼠及貧血大鼠的紅血球細胞。探討非貧血大鼠及貧血大鼠的紅血球細胞膜剛性係數、紅血球細胞的裂解時間、血液的生理條件這三者之間有無關係存在。
由研究結果顯示，貧血大鼠的紅血球細胞膜剛性係數平均為110.909×10-3N/m會明顯大於正常的紅血球細胞膜剛性係數平均為52.785×10-3N/m兩倍左右，而生理條件的RBC、HGB、HCT於貧血時會明顯下降，這兩者皆可用於判定是否貧血的依據，即貧血時紅血球細胞膜剛性係數會變大與生理條件的RBC、HGB、HCT會明顯下降。但紅血球細胞的裂解時間與生理條件以及紅血球細胞膜剛性係數皆無明顯關係，因此不能以紅血球細胞的裂解時間來作為貧血與否的判斷依據。

The purpose of study is investigated to measure the cell membrane stiffness for the red blood cells of anemia and normal rats using AFM. On the other hand, an triangle micro-structure chip is made by micro-manufacture technology then the non-uniform electric field which is worked on the DC pulse voltage to destroy the red blood cells of anemia and normal rats. This study investigated the relationships among cell membrane stiffness, cell lysis time and physiology conditions for the red blood cells of rats.

From the experimental results, that the membrane stiffness of anemia red blood cell, 110.909x10-3N/m, is significantly higher than that of normal red blood cell, 52.785 x10-3N/m. Then the physical conditions of RBC, HGB, HCT in the anemia are decreased significantly, both of them can be used to determine whether the basis for anemia. It means the membrane stiffness of anemia cell increased as the physical conditions of RBC, HGB, HCT decrease. However, the relationship between the cell lysis time of red blood cells and membrane stiffness or physiological conditions is no obvious significance. It is not possible to the cell lysis time of red blood cells whether or not the basis for anemia.

摘要 I
Abstract II
目錄 III
圖表目錄 V
符號說明 VII
第一章 前言 1
1.1 研究動機 1
1.2 研究背景 5
1.3 研究目的 7
第二章 文獻探討 8
2.1 力學分析 8
2.2 以AFM對紅血球細胞之研究 10
2.3 口服腺嘌呤使大鼠慢性腎功能衰竭貧血動物模型的建立 12
2.4 細胞裂解（cell lysis） 13
第三章 理論基礎 15
3.1 AFM簡介 15
3.1.1 AFM基本原理 15
3.1.2 光學感測器 18
3.1.3 探針 20
3.1.4 力—距離曲線量測原理 22
3.1.5 生物樣本楊氏模數（Domke,1998；張,2005）與剛性係數（stiffness）之分析 24
3.2 紅血球 26
3.2.1 紅血球生成 Erythropoiesis 27
3.2.2 紅血球細胞膜 29
3.3 聚二甲基矽氧烷（Polydimethylsiloxane, PDMS） 30
3.4 胞膜電穿孔理論 31
第四章 實驗材料設備與方法 34
4.1 實驗材料 34
4.2 實驗設備簡介 36
4.2.1 JPK Nanowizard AFM簡介 37
4.3 實驗架構與方法 39
4.3.1紅血球生理狀況 41
4.3.2 紅血球細胞樣本製備 41
4.3.3 基板製備 44
4.3.4 血液生理條件分析 45
4.3.5 固定紅血球細胞(李育香,2005) 46
4.3.6 AFM力曲線量測 47
4.3.7 AFM紅血球細胞膜力-距離曲線量測示意圖 51
4.3.8 微影蝕刻製程 52
4.3.9 PDMS注模製程 58
4.3.10 細胞裂解實驗方法與步驟（鄧, 2007） 61
第五章 結果與討論 64
5.1 細胞裂解之分析 64
5.2 生理條件分析 70
5.3 貧血和非貧血大鼠紅血球細胞於液相中之膜剛性係數分析 72
5.4 紅血球細胞膜剛性係數與生理條件之分析 77
5.5 細胞裂解時間與紅血球細胞膜剛性係數的關係 84
5.6 細胞裂解時間與生理條件關係 88
第六章 結論與建議 90
6.1 結論 90
6.2 建議 92
參考文獻 93
附錄一 NO.1紅血球細胞膜剛性係數 97
附錄二 NO.2紅血球細胞膜剛性係數 98
附錄三 NO.3紅血球細胞膜剛性係數 99
附錄四 NO.4紅血球細胞膜剛性係數 100
附錄五 NO.5紅血球細胞膜剛性係數 101
附錄六 NO.6紅血球細胞膜剛性係數 102
附錄七 NO.7紅血球細胞膜剛性係數 103
附錄八 NO.8紅血球細胞膜剛性係數 104
附錄九 NO.9紅血球細胞膜剛性係數 105
附錄十 正常與貧血大鼠的紅血球細胞膜剛性係數（王, 2007） 106
附錄十一 正常與貧血大鼠的生理條件（王, 2007） 107

□ 王威、王箐舟，1999，“腺嘌呤致大鼠慢性腎功能衰竭貧血動物模型的建立”，中國生物制品學雜誌社出版，第十二卷第2期，頁98-99
□ 王政欽，2005，“人類淋巴球白血病活體癌細胞驅動與固定及力學物性之研究”，國立嘉義大學生物機電工程學系研究所，碩士論文
□ 王耀賢，2007，“利用原子力顯微鏡量測正常與貧血大鼠紅血球細胞膜剛性係數之研究”，國立嘉義大學生物機電工程學系研究所，碩士論文
□ 李育香，2005，“以原子力學顯微鏡量測紅血球之奈米力學性質”，國立中興大學物理研究所，碩士論文
□ 呂冠瑩，2005，“3D電極應用於細胞裂解晶片之研究”，國立台灣大學應用力學研究所，碩士論文
□ 何敏夫，1995，血液學，修訂版，合記圖書出版社
□ 吳靖宙、張憲彰，2002， “掃描式探針顯微鏡於生物樣本的量測與應用”，科儀新知，Vol.23，pp.88-99
□ 張佐民，2005， “AFM原子力顯微鏡在生物力學上的研究”，國立清華大學工程科學所，碩士論文
□ 陳登來、陳碧珍，2002，編譯，Atul Metha．Victor Hoffbrand，原著，血液學精義，藝軒圖書出版社，p.9
□ 曾召富，2004，“微幫浦整合於細胞性質量測晶片之設計製作及測試”，國立成功大學醫學工程研究所，碩士論文
□ 鄧志鵬，2007，“利用尖端微結構與直流脈衝裂解細胞之研究”，國立嘉義大學生物機電工程學系研究所，碩士論文
□ Butt, H . J., E. K. Wolff, S. A. C. Gould, B. D. Northern, C. M. Peterson, and P. K. Hansma, 1990, “Imaging cells with the atomicforce microscope”, J. Struct. Biol, 105, 54-61

□ Cai, J., Y. Chen, 2006, “Membrane deformation of unfixed erythrocytes in air with time lapse investigated by tapping mode atomic force microscopy”, Micron, 37, 339–346

□ Cho, Y. H., D. W. Lee, 2007, “A continuous electrical cell lysis device using a low dc voltage for a cell transport andrupture”, Sensors and Actuators B, 124, 84-89

□ Chou, C. F., and F. Zenhausern, 2003, “Electrodeless dielectrophresis for micro total analysis systems”, IEEE engineering in medicine and biology magazine, November/December

□ Domke, J., and M. Radmacher, 1998, “Measure the elastic properties of thin polymer films with the atomic force microscope”, Langmuir, 14, 3320-3325

□ Hofmann, G. A., and G. A. Evans, 1986, “Electronic genetic-physical and biological aspects of cellular electromanipulation”, IEEE Engineering in Medicine and Biology Magazine, 12, 6-25

□ Hutter, J. L., and J. Bechhoefer, 1993, “Calibration of atomic-force microscope tips”, Reviews of Scientific Instruments, 64, 1868-1873
□ Janshoff, A., S. Steltenkamp, C. Rommel, J. Wegener, 2006, “Membrane stiffness of animal cells challenged by osmotic stress”, small, 8-9, 1016-1020

□ Ji, X. L., Y. M. Ma, M. S. Shen, X. Xu, and W. Guan, 2005, “Application of atomic force microscopy in blood research”, World J.Gastroenterol, 1709-1711

□ Kotnik, T., D. Miklavcic, and T. Slivnik,1998, “Time course of transmembrance voltage induced by time-varying electric fields-a method for theoretical analysis and its application”, Bioelectrochemistry and Bioenergetics, 45, 3-16

□ Lee, S. W. ,and Y. C. Tai., 1999, “A micro cell lysis device”, Sensors and Actuators A-Physical, 73(1-2), 74-79

□ Lekka, M., I. Dulinska, M. Targosz, W. Strojny, P. Czuba, W. Balwierz, M. Szymonski, 2006, “Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy”, Journal of biochemical and biophysical methods, 66, 1-11

□ Lu, H., M. A. Schmidt and K. F. Jensen, 2005, “A microfluidic electroporation device for cell lysis”, Lab on a chip, 5, 23-29

□ Nakamura, C., I. Obataya, S. W. Han, N. Nakamura, and J. Miyake, 2005, “Nanoscale operation of a living cell using an atomic force microscope with a nanoneedle”, Nanoletters, 5, 27-30

□ Neumann, E., A. E. Sowers, and C. A. Jordan (Eds), 1989, “ Electroporation and electrofusion in cell biology”, Plenum, 66-72

□ Nowakowski, R., P. Luckham, and P. Winlove, 2001, “Imaging erythrocytes under physiological conditions by atomic force microscope”, Biochimica Biophysica Acta, 1514, 170-17

□ Riethmuller, C., T. E. Schaffer, F. Kienberger, W. Stracke, H. Oberleithner, 2007, “Vacuolar structures can be identified by AFM elasticity mapping”, Ultramicroscopy, 107, 895–901

□ Starodubtseva, M. N., T. G. Kuznetsova, S. A. Chizhik, N. I. Yegorenkov, 2007, “Atomic force microscopy observation of peroxynitrite-induced erythrocyte cytoskeleton reorganization”, Micron, 38, 782–786

□ Takeuchi, M., H. Miyamooto, Y. Sako, H. Komizu, and A. Kusumi, 1998, “Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy”, Biophys. J., 74, 2171-2183

□ Vinckier, A., G. Semenza, 1998, “Measuring elasticity of biological materials by atomic force microscopy”, FEBS letters, 430, 12-16

□ Yokozawa, T., P. H. Zheng, and Oura, et al., 1986, “Animal model of adenine-induce chronic renal failure in rats”, Nephron, 44, 230-234