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系統識別號 U0026-2807201218365300
論文名稱(中文) 探討低強度脈衝超音波刺激對膽固醇操控細胞膜奈米機械力學性質之影響
論文名稱(英文) Effect of Low-Intensity Pulsed Ultrasound Stimulation on Nano-Mechanical Properties of Cholesterol-Manipulated Cell membrane
校院名稱 成功大學
系所名稱(中) 醫學資訊研究所
系所名稱(英) Institute of Medical Informatics
學年度 100
學期 2
出版年 101
研究生(中文) 賴文萱
研究生(英文) Wen-Hsuan Lai
學號 q56991059
學位類別 碩士
語文別 中文
論文頁數 62頁
口試委員 指導教授-王士豪
召集委員-朱銘祥
口試委員-廖峻德
口試委員-吳佳慶
中文關鍵字 超音波  壓深奈米壓痕感測器  膽固醇  機械性質  細胞通透性 
英文關鍵字 low intensity pulsed ultrasound  cell mechanical  nano-indenter 
學科別分類
中文摘要 超音波是一項非常成熟穩定及容易作用的技術,不論是在治療、影像、清洗、刺激…等,其中低強度超音波刺激技術已廣泛應用於各領域,強化欲達成的效果,過去研究顯示低強度超音波的機械振盪能促使細胞產生特性的變化。因此本實驗使用超音波刺激的聲波振盪,觀察低強度超音波對纖維母細胞在回補膽固醇的速度及回補量的影響,並利用光學顯微鏡觀察纖維母細胞受到去除膽固醇及回補時的細胞大小、形態。此外奈米壓痕技術具備簡諧震盪的壓痕探針及精密的荷重與位移感測器,此量測技術能獲得待測物連續接觸剛性與壓痕深度之變化,將用以觀察細胞機械性質的變化。
將細胞加入MβCD去除膽固醇,取30分鐘、1小時、4小時、6小時時間點的細胞,準備進行奈米壓痕的機械性質檢測;回補膽固醇,取同樣時間點的細胞;另一組回補膽固醇的同時,給予強度ISATA50mW/cm2,頻率1MHz,工作週期20%的超音波刺激10分鐘,準備進行奈米壓痕機械性質的檢測。
利用奈米壓痕儀器量測去膽固醇後細胞的機械性質與未去膽固醇前的區別比較:由細胞表面對簡諧接觸剛性與深度關係曲線斜率,代表著細胞膜對簡諧震盪探針在最初20奈米的接觸時的力學反應,進行奈米力學性質的分析,同時以光學顯微鏡觀察細胞形態的改變,兩者結果都與細胞反應相互對應。
英文摘要 The mechanical properties of cells have been verified to correlate to human diseases and aging as well as to various cellular physiological processes, including proliferation, migration, and differentiation. It thus is essential to investigate responses of cells corresponding to chemical and physical stimulations for cellular mechanics studies and other applications. Moreover, measurement of the stiffness of an interested cell is of great importance to comprehend the cellular response with respect to a specific biophysical or biochemical alteration. Hence, current study aims to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on nano-mechanical properties of cholesterol-manipulated cells. The NIH-3T3 fibroblasts were firstly incubated in cholesterol depletion solution. The cells were then insonified by a 1 MHz LIPUS for ten minutes during the process of cholesterol restoration. In situ observation on the variations of living cells adhered on the glass substrate was monitored continuously. The cellular morphological changes were recorded for 8 hours. The nano-mechanical properties of cells were detected with nano-indentation by measuring the cellular membrane of a single cell. The cell properties seemed to be unchanged when adding with serum. On the other hand, the cholesterol depletion tended to lead a majority of cells to retract and become rounding corresponding to the increase of incubation time. The complications may be alleviated with the insonification of LIPUS. The elastic properties of those cholesterol-manipulated cells following LIPUS insonification tended to be improved. This study demonstrates that the variations of nano-mechanical property of cell membrane during cholesterol restoration and LIPUS insonification may be sensitively detected using the depth-sensing nano-indentation technique. The LIPUS insonification may be beneficial to the restoration of elastic properties of cholesterol-manipulated cells.
論文目次 中文摘要 I
ABSTRACT II
誌謝 III
目錄 IV
圖目錄 VIII




第一章 緒論 1
1-1 前言 1
1-2 文獻回顧 5
1-2.1 低強度脈衝超音波刺激 5
1-3 研究目的 12
第二章 理論基礎 14
2-1 超音波 14
2-1.1 波動基本原理 14
2-1.2 波的反射、折射、散射及衰減 16
2-1.3 超音波換器與聲場 18
2-2 細胞簡介 21
2-2.1 細胞基本原理 21
2-2.2 細胞週期 24
2-3 細胞機械性質 25
2-3.1 細胞機械性質及動力傳導 26
2-3.2 細胞力學量測的方法 27
第三章 實驗材料與方法 32
3-1 實驗設計 32
3-2 實驗材料 34
3-2.1 低強度超音波刺激系統 34
3-2.2 纖維母細胞 35
3-2.3 細胞培養 35
3-2.4 藥品配置方法 37
3-2.5 玻璃酸洗 38
3-2.6 細胞去除及回補膽固醇 38
3-2.7 細胞固定 39
3-3 實驗方法 40
3-3.1 超音波刺激 40
3-3.2 壓深奈米壓痕機 41
3-4 分析方法 43
3-4.1 細胞增生分析方法 43
3-4.2 細胞機械性質分析方法 44
3-4.3 統計分析方法 44
第四章 結果與討論 45
4-1 超音波參數 45
4-1.1 能量對細胞生長影響之結果 45
4-2 去除膽固醇對細胞影響之結果 47
4-2.1 細胞形態的變化 47
4-3 回補膽固醇對細胞影響之結果 49
4-3.1 細胞形態的變化 49
4-3.2 細胞機械性質 50
4-4 結果討論 54
第五章 結論 56
5-1 實驗結論 56
5-2 未來展望 57
參考文獻 58
參考文獻 [1]G. Bao and S. Suresh, "Cell and Molecular Mechanics of Biological Materials," Nature Materials, vol. 2, pp. 715-725, 2003.
[2]S. Park, D. Koch, R. Cardenas, J. Käs, and C. K. Shih, "Cell Motility and Local Viscoelasticity of Fibroblasts," Biophysical Journal, vol. 89, pp. 4330-4342, 2005.
[3]G. Y. H. Lee and C. T. Lim, "Biomechanics Approaches to Studying Human Diseases," Trends in Biotechnology, vol. 25, pp. 111-118, 2007.
[4]S. E. Cross, Y.-S. Jin, J. Rao, and J. K. Gimzewski, "Nanomechanical Analysis of Cells from Cancer Patients," Nature Nanotechnology, vol. 2, pp. 780-783, 2007.
[5]M. N. Starodubtseva, "Mechanical Properties of Cells and Ageing," Ageing Research Reviews, vol. In Press.
[6]R. Matzke, K. Jacobson, and M. Radmacher, "Direct, High-Resolution Measurement of Furrow Stiffening during Division of Adherent Cells," Nature Cell Biology, vol. 3, pp. 607-610, 2001.
[7]V. M. Laurent, S. Kasas, A. Yersin, T. E. Schäfer, S. Catsicas, G. Dietler, A. B. Verkhovsky, and J.-J. Meister, "Gradient of Rigidity in the Lamellipodia of Migrating Cells Revealed by Atomic Force Microscopy," Biophysical Journal, vol. 89, pp. 667-675, 2005.
[8]Lee G Y H and Lim C T 2007 Trends Biotechnol.
[9]I. Titushkin and M. Cho, "Modulation of Cellular Mechanics during Osteogenic Differentiation of Human Mesenchymal Stem Cells," Biophysical Journal, vol. 93, pp. 3693-3702, 2007.
[10]K. A. Addae-Mensah and J. P. Wikswo, "Measurement Techniques for Cellular Biomechanics In Vitro," Experimental Biology and Medicine, vol. 233, pp. 792-809, July 1, 2008 2008.
[11]M. Radmacher, "Studying the Mechanics of Cellular Processes by Atomic Force Microscopy," in Methods in Cell Biology. vol. 83: Academic Press, 2007, pp. 347-372.
[12]E. K. Dimitriadis, F. Horkay, J. Maresca, B. Kachar, and R. S. Chadwick, "Determination of Elastic Moduli of Thin Layers of Soft Material Using the Atomic Force Microscope," Biophysical Journal, vol. 82, pp. 2798-2810, 2002.
[13]L. Sirghi and F. Rossi, "Adhesion and Elasticity in Nanoscale Indentation," Applied Physics Letters, vol. 89, pp. 243118-3, 2006.
[14]Gail ter, Haar, “Therapeutic ultrasound,” European Journal of ultrasound, Vol. 9,
No. 1, pp. 3-9, 1999.
[15]P. Reher, E. N. I. Elbeshir, W. Harvey, S. Meghji and M. Harris, “The stimulation of bone formation in vitro by therapeutic ultrasound,” Ultrasound in Medicine & Biology, Vol. 23, No. 8, pp. 1251-1258, 1997.
[16]W. Y. Shi, M. Schafer, S. Dubin, M. O'Connor, C. Ozturk, M. C. Chou, Skin temperature and impedance measurement in ultrasound wound treatment,” IEEE Biomedical Engineering Conference, No.13, pp. 348-350, 1996.
[17]J. J. Kaufman, H. Popp, A. Chiabrea, and A. A. Pilla, “The effect of ultrasound on the electrical impedance of biological cells,” IEEE engineering in medicine & biology society 10th annual international conference, Vol. 2, No. 6, pp. 753-754,1988.
[18]M. Dyson, “Non-thermal cellular effects of ultrasound,” Britizn Journal of Cancer- Supplement, Vol. 45, No. 5, pp. 165-171, 1982.
[19]“Other nonthermal bioeffects: organs, cells and tissues,” Ultrasound in Medicine & Biology, Vol. 24, Supplement 1, pp. S35-S39, 1998.
[20]M. Radmacher, "Studying the Mechanics of Cellular Processes by Atomic Force Microscopy," in Methods in Cell Biology. vol. 83: Academic Press, 2007, pp. 347-372.
[21]K. A. Addae-Mensah and J. P. Wikswo, "Measurement Techniques for Cellular Biomechanics In Vitro," Experimental Biology and Medicine, vol. 233, pp. 792-809, July 1, 2008 2008.
[22]X. Li and B. Bhushan, "A Review of Nanoindentation Continuous Stiffness Measurement Technique and Its Applications," Materials Characterization, vol. 48, pp. 11-36, 2002.
[23]M. Dyson, J. B. Pond, J. Joseph and R. Warwick, “The stimulation of tissue regeneration by means of ultrasound,” Clinical Science, Vol. 35, No. 2,pp. 273-285, 1968.
[24]S. R. Young and M. Dyson, “Macrophage responsiveness to therapeutic ultrasound,” Ultrasound in Medicine & Biology, Vol. 16, No. 8, pp. 809-816,1990.
[25]M. Schafer, S. Dubin, A. Geshury, and F. Ko, “Experimental methods for ultrasonically enhanced wound healing,” IEEE Ultrasonics Symposium, Vol. 3,No. 5, pp. 1853-1856, 1994.
[26]J. J. Kaufman, H. Popp, A. Chiabrea, and A. A. Pilla, “The effect of ultrasound on the electrical impedance of biological cells,” IEEE engineering in medicine &biology society 10th annual international conference, Vol. 2, No. 6, pp.753-754,1988.
[27]L. O. Kober and J. W. Ellwart, “Effect of pulse length of ultrasound on cell membrane damage in vitro,” Journal of the Acoustical Society of America, Vol.86, No. 1, pp. 6-7, 1989.
[28]S. R. Young and M. Dyson , “Effect of therapeutic ultrasound on healing of full-thickness excised skin lesions,” Ultrasonics, Vol. 28, No. 3,pp. 175-180,1990
[29]L. Maxwell, T. Collecutt, M. Gledhill, S. Sharma, S. Edgar and J. B. Gavi “The augmentation of leucocyte adhesion to endothelium by therapeutic ultrasound,” Ultrasound in Medicine & Biology, Vol. 20, No. 4, pp.383-390, 1994.
[30]P. G De Deyne, M. Kirsch - Volders, “In vitro effects of therapeutic ultrasound on the nucleus of human fibroblasts,” Physical Therapy, Vol. 75, No. 7,pp. 629-634, 1995.
[31]A. Ramirez, Jeams. A. Schwane, C. Mcfarland, and B. Starcher, “The effect of ultrasound on collagen synthesis and fibroblast proliferation in vitro,”Medicine & Science in Sports & Exercise, Vol. 29, No. 3, pp. 326-332, 1997.
[32]P. Reher, E. N. I. Elbeshir, W. Harvey, S. Meghji and M. Harris, “The stimulation of bone formation in vitro by therapeutic ultrasound,” Ultrasound in Medicine & Biology, Vol. 23, No. 8, pp. 1251-1258, 1997.
[33]I. Hrazdira, J. Skorpikova, M. Dolnikova,“Ultrasonically induced alterations of cultured tumour cells,” European Journal of ultrasound, Vol. 8, No. 1, pp.43-49, 1998.
[34]T. Kokubu, N. Matsui, H. Fujioka, M. Tsunoda, and K. Mizuno, “Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of Cyclooxygenase - 2 mRNA in mouse osteoblasts,” Biochemical and Biophysical Research Communications, Vol. 256, No. 2, pp. 284-287, 1999.
[35]M. Ito, Y. Azuma, T. Ohta, and K. Komoriya, “Effects of ultrasound and 1,25-Dihydroxyvitamin D3 on growth factor secretion in co-cultures of osteoblasts and endothelial cells,” Ultrasound in Medicine & Biology, Vol. 26, No. 1, pp.161-166, 2000.
[36]Y.-L. Wang and D. S. Discher, Cell Mechanics. Boston: Elsevier Academic Press, 2007.
[37]J. Wolff, The Law of Bone Remodeling. Berlin Heidelberg New York: Springer, 1986.
[38]Y. D. Yang “Continuous Depth-sensing Nano-mechanical Characterization of Living, Fixed and Dehydrated Cells Attached on a Glass Substrate,” Nanotechnology, 2010.
[39]J. R. Withers and D. E. Aston, "Nanomechanical Measurements with AFM in the Elastic Limit," Advances in Colloid and Interface Science, vol. 120, pp. 57-67, 2006.
[40]G. Y. H. Lee and C. T. Lim, "Biomechanics Approaches to Studying Human Diseases," Trends in Biotechnology, vol. 25, pp. 111-118, 2007.
[41]K. A. Addae-Mensah and J. P. Wikswo, "Measurement Techniques for Cellular Biomechanics In Vitro," Experimental Biology and Medicine, vol. 233, pp. 792-809, July 1, 2008 2008.
[42]V. M. Laurent, S. Kasas, A. Yersin, T. E. Schäfer, S. Catsicas, G. Dietler, A. B. Verkhovsky, and J.-J. Meister, "Gradient of Rigidity in the Lamellipodia of Migrating Cells Revealed by Atomic Force Microscopy," Biophysical Journal, vol. 89, pp. 667-675, 2005.
[43]I. Titushkin and M. Cho, "Modulation of Cellular Mechanics during Osteogenic Differentiation of Human Mesenchymal Stem Cells," Biophysical Journal, vol. 93, pp. 3693-3702, 2007.
[44]A. L. Weisenhorn, M. Khorsandi, S. Kasas, V. Gotzos, and H.-J. Butt, "Deformation and Height Anomaly of Soft Surfaces Studied with an AFM," Nanotechnology, vol. 4, p. 106, 1993.
[45]M. Radmacher, "Measuring the Elastic Properties of Biological Samples with the AFM " IEEE Engineering in Medicine and Biology Magazine, vol. 16, pp. 47-57, 1997.
[46]D. A. Christensen, Ultrasonic Bioinstrumentations, John. Wiley & Sons, 1988.
[47]K. Kirk Shung and M. Zipparo, “Ultrasonic transducers and arrays,” IEEE engineering in medicine and biology, Vol. 15, No. 6, pp. 20-30, 1996.
[48]K. Kirk Shung, “Diagnostic ultrasound: Imaging and Blood Flow Measurements,” Boca Raton, FL:CRC Press, 2006.
[49]B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter, Molecular Biology of the Cell, 4th ed. New York: Garland Science, 2002.
[50]W. C. Oliver and G. M. Pharr, "An Improved Technique for Determining Hardness and Elastic Modulus Using Load and Displacement Sensing Indentation Experiments," Journal of Materials Research, vol. 7, pp. 1564-1583, 1992.
[51]D. M. Ebenstein and L. A. Pruitt, "Nanoindentation of Soft Hydrated Materials for Application to Vascular Tissues," Journal of Biomedical Materials Research A, vol. 69, pp. 222-232, 2004.
[52]C. T. Lim, E. H. Zhou, and S. T. Quek, "Mechanical Models for Living Cells--a Review," Journal of Biomechanics, vol. 39, pp. 195-216, 2006.
[53]D. M. Ebenstein and L. A. Pruitt, "Nanoindentation of Biological Materials," Nano Today, vol. 1, pp. 26-33, 2006.
[54]S. H. Chen, C. Y. Chiu, and S. H. Wang, “The growth of Osteoblasts stimulated by various time duration of low intensity pulsed ultrasounds,” IEEE Conference, 365-368, 2009
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