簡易檢索 / 詳目顯示

回結果列表
研究生： 黃佩瑜
Huang, Pei-Yu
論文名稱： 以原子力顯微術在病毒感染細胞之形態觀察活體細胞量測技術之發展
Morphological Observation for Virus-Infected Cells and the Measuring Technology Developed Based on Atomic Force Microscopy
指導教授： 張憲彰
Chang, Hsien-Chang
學位類別： 碩士
Master
系所名稱： 工學院 - 醫學工程研究所
Institute of Biomedical Engineering
論文出版年： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 76
中文關鍵詞： 原子力顯微術 、病毒感染宿主與非宿主細胞 、活體細胞量測
外文關鍵詞： living cell measuring technology, virus infected host /non-host cell, AFM
相關次數： 點閱：91  下載：1 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 　　原子力顯微術（AFM）除具有極佳的高解析度與簡單的樣本前處理之外，因也可在液相中取像，已有許多文獻用來觀察微生物、細胞的微結構，亦有藉之掃描到病毒感染細胞後其表面的出芽模形態，甚至用以探討某些病毒外套膜的表面特徵。本研究首先，以兼嗜性野生型小鼠白血病病毒（MMLV），分別去感染人類膀胱癌細胞（J82）、人類肺癌細胞（A549）及老鼠纖維母細胞（NIH3T3）等三種宿主細胞，對照性地再以親嗜性MMLV進行宿主與非宿主細胞受感染6 hr後之細胞膜表面的觀察，黏貼於培養皿上的細胞樣本，先以福馬林固定及磷酸鹽緩衝液、去離子水清洗，繼而在空氣中自然乾燥後進行AFM掃描。結果顯示，當NIH3T3細胞受上述兩種MMLV感染時，均在其表面上發現許多可能是病毒融合或細胞胞飲作用所造成的孔洞。這在J82及A549細胞受兼嗜性MMLV感染時亦被發現，然若是受親嗜性MMLV感染的話，則無此類似的現象。其次，我們也將上述的親嗜性MMLV與其經外套膜蛋白上修飾Arg-Gly-Asp（RGD）的MMLV，分別探討其對倉鼠口腔癌細胞（HCDB-1）、人類微血管表皮細胞（HMEC-1）兩種非宿主細胞，以及NIH3T3宿主細胞感染能力的影響。其結果得知，受RGD修飾之MMLV感染的HCDB-1、HMEC-1，亦會出現和NIH3T3之細胞膜表面上類似的凹陷及孔洞現象，顯示經修飾後的病毒可能已轉變成可對非宿主細胞具感染作用。最後，為要建立液相中病毒感染細胞的過程的觀察可能性，我們也嘗試了在液相中活體細胞表面的掃描技術探討，並藉儀器上的力調變顯微術紀錄與力/位移曲線，量測整個細胞表面軟硬的分佈。經分析細胞表面各點的力學特性，由楊氏係數求取得知，細胞核中心部位為其周圍區域的0.05倍。

    　　Atomic force microscopy (AFM) has a high resolution, easy sample preparation and has the benefit to get the images under the real physiological condition. It has been used to explore a wide variety of biological samples, including microorganism, viruses and cells, etc. Previous studies have shown that AFM can be used to detect the surface features of viral envelope protein and the normal budding of wild-type virions from cell surfaces. In this study, we attempt to observe the morphological changes of the cells surface after wild-type Moloney murine leukemia virus(MMLV) infection. The above results we obtained were all scanning mode after drying the sample. To understand the infectious phenotype of the ecotropic as well as amphotropic MMLV infection, the morphological of J82, A549 and NIH3T3 cells were compared before and after the viral infection by AFM. The results showed that the cellular features of NIH3T3 cells were different from J82 and A549. The surface feature of NIH3T3 cells after viral infection exhibited the hollow formation however the J82 and A549 did not. This result suggested that the hollow formation might be caused by the fusion of virus into cell membrane or pinocytosis of cell with virus. Otherwise, we tried to a RGD sequence was inserted into the envelope protein MMLV and observe the ability of viral infection in non-host cells. The HCDB-1, HMEC-1, and NIH3T3 cells infected by modified MMLV were observed the morphological by AFM. The results showed that infection of those cells by using modified viruses resulted in indentation formation of the cell surface in all cells examined. Besides, we have established a living cell measuring technique by AFM and qualitatively and quantitatively measured the elastic properties of cell surface. Development of this measuring technique in liquid phase offers an easy way to investigate the changes of cellular structures in virus-host cell interactions during infection processes in the future.

    摘要.............I Abstract.............II 致謝.............III 目錄.............IV 表目錄.............VIII 圖目錄.............IX 第一章 緒論.............1 1.1 研究背景.............1 1.2 原子力顯微術的原理.............2 1.2.1 原子力顯微鏡的量測模式.............3 1.2.2 原子力顯微鏡的成像模式.............5 1.2.3 原子力顯微鏡硬體架構.............6 1.2.4 提高原子力顯微術影像的方法.............7 1.2.5 原子力顯微術的應用.............9 1.3 原子力顯微術在生物樣本的應用.............11 1.3.1 原子力顯微術在生物樣本觀察.............11 1.3.2 病毒形態觀察.............11 1.3.3 病毒感染細胞觀察.............12 1.3.4 修飾觀察.............13 1.3.5 細胞觀察.............14 1.3.6 細胞力學觀察.............16 1.3.7 原子力顯微術在生物應用上的展望.............17 1.4 本研究中之病毒、細胞的基本特性.............18 1.4.1 小鼠白血病病毒的基本特性.............18 1.4.2 病毒與宿主細胞的交互作用.............19 1.4.3 病毒感染細胞的階段過程.............20 1.4.4 病毒感染細胞時的表面變化.............21 1.4.5 病毒感染細胞的分析.............22 1.5 本研究之目的.............24 1.5.1 病毒感染細胞的觀察.............24 1.5.2 形態學解釋感染現象.............25 1.6 本實驗研究架構圖.............26 第二章 實驗設備與方法.............27 2.1 實驗硬體設備與實驗材料.............27 2.1.1 儀器設備與操作.............27 2.1.2 實驗材料.............29 2.2 AFM掃描細胞與病毒的影像.............31 2.2.1 AFM掃描模式與方法.............31 2.2.2 探針的選用.............32 2.2.3 固定液的選用.............33 2.3 小鼠白血病病毒（Moloney murine leukemia virus）.............35 2.3.1 小鼠白血病病毒的結構.............35 2.3.2 小鼠白血病病毒的製造.............36 2.3.3 小鼠白血病病毒表面特徵.............36 2.4 Moloney murine leukemia virus感染細胞.............38 2.4.1 病毒感染宿主與非宿主細胞的影響.............38 2.4.2 Wt-MMLV感染J82、A549和NIH3T3細胞觀察.............38 2.4.3 MMLV感染HCDB-1、HMEC-1和NIH3T3細胞觀察.............39 2.5 原子力顯微術觀察液相活體細胞技術之建立.............41 2.5.1 AFM之液相活體量測方法.............41 2.5.2 AFM之力學分析.............42 2.5.3 生化物質的黏滯性量測.............43 2.5.4 楊氏係數之求取.............44 第三章 結果與討論.............46 3.1 原子力顯微術影像的解析.............46 3.2 原子力顯微術掃描MMLV的影像.............48 3.1.1 Wild-type MMLV的製造.............48 3.1.2 Ecotropic、Amphotropic和Modified MMLV影像.............48 3.2 Wt-MMLV感染人類膀胱癌細胞.............50 3.2.1 J82細胞未感染影像.............50 3.2.2 Ecotropic MMLV感染J82細胞後影像分析.............50 3.2.3 Amphotropic MMLV感染J82細胞後影像分析.............51 3.2.4 MMLV感染J82細胞表面的變化.............51 3.3 Wt-MMLV感染人類肺癌細胞.............53 3.3.1 A549細胞未感染影像.............53 3.3.2 Ecotropic MMLV感染A549細胞後影像分析.............53 3.3.3 Amphotropic MMLV感染A549細胞後影像分析.............54 3.3.4 MMLV感染A549細胞表面的變化.............54 3.4 Ecotropic和Modified MMLV感染倉鼠口腔癌細胞.............56 3.4.1 HCDB-1細胞未感染影像.............56 3.4.2 Ecotropic MMLV感染HCDB-1細胞後影像分析.............56 3.4.3 Modified MMLV感染HCDB-1細胞後影像分析.............57 3.4.4 MMLV感染HCDB-1細胞表面的變化.............57 3.5 Ecotropic和Modified MMLV感染人類微血管內皮細胞.............59 3.5.1 HMEC-1細胞未感染影像.............59 3.5.2 Ecotropic MMLV感染HMEC-1細胞後影像分析.............59 3.5.3 Modified MMLV感染HMEC-1細胞後影像分析.............60 3.3.4 MMLV感染HMEC-1細胞表面的變化.............60 3.6 MMLV感染鼠纖維母細胞.............62 3.6.1 NIH3T3細胞未感染影像.............62 3.6.2 Ecotropic MMLV感染NIH3T3細胞後影像分析.............62 3.6.3 Amphotropic MMLV感染NIH3T3細胞後影像分析.............63 3.6.4 Modified MMLV感染NIH3T3細胞後影像分析.............63 3.6.5 MMLV感染NIH3T3細胞表面的變化.............63 3.7 AFM液相活體細胞量測.............65 3.7.1 細胞形態.............65 3.7.2 細胞力學分析.............65 第四章 結論.............68 4.1 病毒與細胞形態觀察.............68 4.2 原子力顯微術量測液相活體細胞技術之評估.............70

    1. 沈萍,汪蕙蘭,“微生物學”, 五南圖書出版公司, 2003
    2. 楊繼江,“病毒學概論”, 藝軒圖書出版社, 2003
    3. 林錦泉,王譽朝,“原子力顯微鏡在生物科技上的應用”, 生物科技, 14, 2003
    4. M.L. Ng, J.W.M. Lee, M.L.N. Leong, A. E. Ling, H. C. Tan, E. E. Ooi, “Topographic changes in SARS coronavirus–infected cells during late stages of infection”, Emerging Infectious Diseases, 10, 2004, 1907-1914
    5. G. Binning, H. Rohrer, “Scanning tunneling microscopy from birth to adolescence”, Rev. Modern Physics, 59, 1987, 615-619
    6. M. A. Poggi, E. D. Gadsby, L. A. Bottomley “Scanning probe microscopy”, Anal. Chem., 76, 2004, 3429-3444
    7. G. Binning, C. F. Quate, C. Gerber, “Atomic force microscope”, Phys. Rev. Lett., 56, 1986, 930-933
    8. V. J. Morris, A. R. Kirby, A. P Gunning, “Atomic force microscopy for biologists”, Imperial College Press: London, 1999
    9. Park Scientific Instruments Corp., Users Guide to Autoprobe CP, Part II, http://www.park.com.
    10. NT-MDT Corp., SPM introduction, http://www.ntmdt.ru
    11. Digital Instruments Corp., Data Sheets, http://www.di.com
    12. 吳靖宙,張憲彰,“掃描式探針顯微鏡於生物樣本的量測與應用”, 科儀新知23, 2002, 88-98
    13. I. Obataya, C. Nakamura, S. Han, J. Miyake, “Mechanical sensing of the penetration of various nanoneedles into a living cell using AFM”, Biosens. Bioelectron., 20, 2005, 1652-1655
    14. S. Sheng, D. M. Czajkowsky, Z. Shao, “AFM tips: how sharp are they?”, J. Microsc., 196, 1999, 1-5
    15. Biological Applications of SPM , www.spmtips.com/bibliography/biology
    16. W. F. Kolbe, D. F. Ogletree, M. B. Salmeron, “Atomic force microscopy imaging of T4 bacteriophages on silicon substrates”, Ultramicroscopy, 42, 1992, 1113-1117.
    17. N. Matsko, D. Klinov, A. Manykin, V. Demin, S. Klimenko, “Atomic force microscopy analysis of bacteriophages phiKZ and T4,” J. Electron Microsc. , 50, 2001, 417-422
    18. Y. G. Kuznetsov, A. Low, H. Fan, McPherson,“ Atomic force microscopy imaging of retroviruses: human immunodeficiency virus and murine leukemia virus”, Scanning, 26, 2004, 209-216
    19. Y. G. Kuznetsov, S. Datta, N.H Kothari, A. Greenwood, H. Fan, A. McPherson, “Atomic force microscopy investigation of fibroblasts infected with wild-type and mutant murine leukemia virus (MuLV)”, Biophys. J., 83, 2002, 3665-3674
    20. W. Haberle, J. K. Horber, F, Ohnesorge, D.P. Smith, G. Binnig, “In situ investigations of single living cells infected by viruses”, Ultramicroscopy, 43, 1992, 1161-1167
    21. J. W. Lee, M. L. Ng, “A Nano-view of West Nile Virus-induced Cellular Changes during Infection”, Nanobiotechnology, 2(1), 2004
    22. M. Moloney, L. M.Donnell, H. O. Shea, “Immobilisation of Semliki Forest virus for atomic force microscopy”, Ultramicroscopy, 91, 2002, 275-279
    23. M. D. Palma, M. A. Venneri, L. Naldini, “In vivo targeting of tumor endothelial cells by systemic delivery of lentiviral vectors”, Human Gene Therapy, 14, 2003, 1193-1206
    24. Y. G. Kuznetsov, A. Low, H. Fan, A. M. Pherson, “Atomic force microscopy investigation of wild-type Moloney murine leukemia virus particles and virus particles lacking the envelope protein”, Virology, 323, 2004, 189-196
    25. E. Lesniewska, M. C. Giocondi, V. Vie, E. Finot, J.P. Goudonnet, C. L. Grimellec., “Atomic force microscopy of renal cells: limits and prospects”, Kidney Int. Suppl., 65, 1998, 42-48
    26. L. I. Pietrasanta, A. Schaper, T. M Jovin, “Imaging subcellular structures of rat mammary carcinoma cells by scanning force microscopy”, Cell Sci., 107, 1994, 2427-2437
    27. H. Haga, S. Sasaki, K. Kawabata, E. Ito, T. Ushiki, T. Sambongi., “Elasticity mapping of living fibroblasts by AFM and immunofluorescence observation of cytoskeleton”, Ultramicroscopy, 82, 2000, 253-258
    28. D. Han, W. Ma, F. Liao, M. Yeh, Z. Ouyang, Y. Sun, “Time-series observation of the spreading out of microvessel endothelial cells with atomic force microscopy”, Phys. Med. Biol., 48, 2003, 3897-3909,
    29. F. Braet , D. Vermijlen, V. Bossuyt, R. D. Zanger, E. Wisse, “Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope”, Ultramicroscopy, 89, 2001, 265-273
    30. N. J. Tao, N. M. Lindsay, S. Lees. “Relative microelastic mapping of living cells by AFM” Biophys. J., 74, 1998, 1564-1578
    31. Y. C. Fung, “Biomechanics –mechanics properties of living tissues”, Springer, New York, 1993
    32. A. L. Weisenhorn, M. Khorandi, “Deformation and height anomaly of soft surfaces studied with the AFM”, Nanotechnology, 4, 1993, 106-113
    33. M. Grandboisa, W. Dettmannb, M. Benoitb, H. E. Gaubb, “Affinity imaging of red blood cells using an atomic force microscope” Histochemistry and Cytochemistry, 48, 2000, 719-724,
    34. F. M. Ohnesorge, J. K. Horber, W Haberle, C. P. Czerny, D.P. Smith, G Binnig, “AFM review study on pox viruses and living cells”, Biophys. J., 73, 1997, 2183-2194
    35. D. Knebel, M. Amrein, Voigt K, Reichelt R, “Scanning force microscopy at the air-water interface of an air bubble coated with pulmonary surfactant”, Biophys. J., 82, 2002, 474-480
    36. B. Weyn, W. Kalle, S. K. Singh, E. V Marck, H. Tanke, W. J. Jacob, “Atomic force microscopy: influence of air drying and fixation on the morphology and viscoelasticity of cultured cells”, J. Microsc. 189, 1998, 172-180
    37. A. Vinckier, G. Semenza, “Measuring elasticity of biological materials by atomic force microscopy”, FEBS Lett., 430, 1998, 12-16
    38. H. X. You, L. Yu, “AFM imaging of living cells: progress, problems and prospects”, Methods Cell Sci., 21, 1999, 1-17
    39. H. Zimmermann, R. Hagendorn, E. Richter, G. Fuhr, “Topography of cell traces stuidied by atomic force miceoscopy”, Biophys. Lett., 28, 2003, 516-525
    40. P. D. Robbins, S. C. Ghivizzani, “Viral vector for gene therapy,” Pharmacology Therapy, 80, 1998, 35-47
    41. D. B. Hen,“Gene therapy principle and practice”, 九州圖書文物有限公司, 1995
    42. N. J. Dimmock, S. B. Primrose著, 張信之編譯, “現代病毒學大綱”, 藝軒圖書出版社, 1989
    43. 陳蘭君, “一般病毒學精華”, 合記圖書出版社, 1995
    44. 瞿中和,王喜中,“細胞分子生物學”, 九州圖書文物有限公司, 2001
    45. 劉武哲, “醫用病毒學及其研究方法”, 藝軒圖書出版社, 1996
    46. 林良平, “微生物顯微鏡學, Microscopy for microorganisms”, 藝軒圖書出版社, 2002
    47. Vecco Instruments Corp., SPM introduction, http://www.vecco.com
    48. M. Moloney, L. M. Donnell, H.O. Shea., “Atomic force microscopy of BHK-21 cells: an investigation of cell fixation techniques”, Ultramicroscopy, 100, 2004, 153-161
    49. GenScript Vector-based siRNA Protocol (Lenti-viral), Technical Manual No. 0175
    50. 林鶴南,李龍正,劉克迅, “原子力顯微術於奈米加工之應用” , 科儀新知, 17, 1995, 29-38
    51. Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Dennis Bray, Karen Hopkin, Keith Roberts, Peter Walter, “Essential cell biology”, Second edition, Garland Science

    下載圖示 校內：2006-07-19公開
    校外：2006-07-19公開
    QR CODE