AFM球形针尖的制作及癌细胞力学特性的实验研究
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摘要
癌症,近几十年来已对人类健康构成强大威胁,虽然对于肿瘤的组织病理学诊断和放化疗技术日趋成熟,但癌症的发病机理和转移机制等根本性问题却尚未明晰。利用AFM在单细胞水平对癌细胞进行相关分析已成为当今癌症研究的最新热点,具有其它技术无法比拟的优势,但大多集中于AFM形貌成像等基础研究,从机械角度出发对于癌细胞的力学特性进行分析限于研究手段等原因却受到较少关注,而力学特性与癌细胞转移过程息息相关。本课题正是利用自行制作的小球探针,基于AFM的Nanoindentation功能在液态环境下对癌细胞的力学特性进行测定,分析其对转移的影响。
首先,对现有的应用于固体纳米压的痕三角形悬臂探针针尖进行功能化改造,扩展了AFM在生物领域的应用。利用实验室现有设备,自行制作出满足液态下对活细胞进行纳米压痕的小球探针,并利用Cleveland方法对其弹性常数进行了校准。该制作过程同样适用于其它微米级球状物向微米级尖状物的粘附。
然后,分析了液态环境下实施活细胞纳米压痕实验所涉及的各方面条件,包括活细胞的选择、培养、转运,液态实验环境建立,仪器的改装,实验的改进,操作的优化,以及具体实验前仪器的标定等等,每一项都关乎实验能否顺利进行。其中自行总结的液态下压痕目标细胞快速定位方法具有很好的实用性和通用性。
最后,在保持细胞最佳生理活性的液态环境下,对转移性不同的Anip-973和AGZY-83a两种肺癌细胞进行AFM纳米压痕实验,并利用Hertz接触方程及应力松弛模型对实验结果进行非线性拟合,再通过方差分析比对力学特性参数结果,以研究其对癌变转移的影响。
Cancer has posed a powerful threat to human health in recent decades. The histopathological diagnosis and chemotherapy technology of the tumor is maturing; however, fundamental issues as the pathogenesis of cancer and metastatic mechanism stay vague. As the latest hot spot researching cancer cells in the single-cell level based AFM, the technology can not match advantage, but most focused on basic research, e.g. the AFM topography imaging. The method analyzing mechanical properties of cancer cells from a mechanical view has been limited by investigating way, which has been less reason for concern, are closely related with cancer cells metastasis. The paper aimed to research their mechanical properties and the impact of metastasis in the use of self-made spherical ball tip, based on the AFM nanoindentation features in the liquid environment.
First, functional transformation of existing solid triangle cantilever probe tip used in solid namoindentation, extended the application of AFM in the biological field. By laboratory equipment existing, fabricated spherical tip fitting for nanoindentation of living cells in liquid by self, and calibrated its elastic constants by Cleveland method. The production process also applies to stick other micron ball to the micron spikes.
Then, analyzed of all aspects of the conditions involved in the nanoindentation of living cells in the liquid environment, including the choice of living cells, culture, transport, liquid experiment environment to establish, what’s more, the modification of the equipment, test improvement, operation optimization, and the specific calibration of the instrument before the experiment, etc., each of which relates to experiment can proceed smoothly. The summary of quick cell targeting in liquid indentation has good practicality and versatility.
Finally, in the PBS liquid environment to maintain their best physical activity, experiments were performed between Anip-973 and AGZY-83a lung cancer cells with different metastatic ability. The results were implemented nonlinear fitting upon the stress-relaxation model derived from Hertz contact equation. ANOVA was conducted to determine the differences between each of the mechanical parameters, in order to examine its impact on cancer metastasis.
首先,对现有的应用于固体纳米压的痕三角形悬臂探针针尖进行功能化改造,扩展了AFM在生物领域的应用。利用实验室现有设备,自行制作出满足液态下对活细胞进行纳米压痕的小球探针,并利用Cleveland方法对其弹性常数进行了校准。该制作过程同样适用于其它微米级球状物向微米级尖状物的粘附。
然后,分析了液态环境下实施活细胞纳米压痕实验所涉及的各方面条件,包括活细胞的选择、培养、转运,液态实验环境建立,仪器的改装,实验的改进,操作的优化,以及具体实验前仪器的标定等等,每一项都关乎实验能否顺利进行。其中自行总结的液态下压痕目标细胞快速定位方法具有很好的实用性和通用性。
最后,在保持细胞最佳生理活性的液态环境下,对转移性不同的Anip-973和AGZY-83a两种肺癌细胞进行AFM纳米压痕实验,并利用Hertz接触方程及应力松弛模型对实验结果进行非线性拟合,再通过方差分析比对力学特性参数结果,以研究其对癌变转移的影响。
Cancer has posed a powerful threat to human health in recent decades. The histopathological diagnosis and chemotherapy technology of the tumor is maturing; however, fundamental issues as the pathogenesis of cancer and metastatic mechanism stay vague. As the latest hot spot researching cancer cells in the single-cell level based AFM, the technology can not match advantage, but most focused on basic research, e.g. the AFM topography imaging. The method analyzing mechanical properties of cancer cells from a mechanical view has been limited by investigating way, which has been less reason for concern, are closely related with cancer cells metastasis. The paper aimed to research their mechanical properties and the impact of metastasis in the use of self-made spherical ball tip, based on the AFM nanoindentation features in the liquid environment.
First, functional transformation of existing solid triangle cantilever probe tip used in solid namoindentation, extended the application of AFM in the biological field. By laboratory equipment existing, fabricated spherical tip fitting for nanoindentation of living cells in liquid by self, and calibrated its elastic constants by Cleveland method. The production process also applies to stick other micron ball to the micron spikes.
Then, analyzed of all aspects of the conditions involved in the nanoindentation of living cells in the liquid environment, including the choice of living cells, culture, transport, liquid experiment environment to establish, what’s more, the modification of the equipment, test improvement, operation optimization, and the specific calibration of the instrument before the experiment, etc., each of which relates to experiment can proceed smoothly. The summary of quick cell targeting in liquid indentation has good practicality and versatility.
Finally, in the PBS liquid environment to maintain their best physical activity, experiments were performed between Anip-973 and AGZY-83a lung cancer cells with different metastatic ability. The results were implemented nonlinear fitting upon the stress-relaxation model derived from Hertz contact equation. ANOVA was conducted to determine the differences between each of the mechanical parameters, in order to examine its impact on cancer metastasis.
引文
[1] Jemal A.,Tiwari R.C.,Murray T. Cancer Statistics,2004[J]. CA Cancer J Clin,2004,54(1):8-29.
[2]袁倬斌.有机分析研究进展—单原子、单分子、单细胞的分析进展[J].分析实验室,2002,21(1):97-104.
[3] Gabriel Y.,Chwee T. Biomechanics Approaches to Studying Human Diseases[J]. Trends in Biotechnology,2007,25(3):111-118.
[4] Sergey N. Single-cell Analysis Avoids Sample Processing Bias[J]. Journal of Chromatography B,2000,741(1) :31~35.
[5] Benjamin A. Cellular Biomechanics Investigated by Atomic Force Microscopy[D]. Montreal:Thesis Submitted to McGill University for the Degree of Doctor,2004:4-6.
[6]张经济.追踪癌细胞转移之谜[J].中国医药指南,2003,05:48-49.
[7] Hanahan D.,Weinberg R. The Hallmarks of Cancer[J]. Cell,2000,100(1):57-70.
[8] Susana M.,Rafael B. Stress Relaxation Microscopy: Imaging Local Stress in Cells[J]. Journal of Biomechanics,2010,43(1):349-354.
[9]胡德红,龚萍,蔡林涛.纳米技术在癌症早期诊断和治疗中的研究与展望[J].先进技术研究通报,2009,03 (01):7~12.
[10]张汝冰,刘宏英,李凤生.纳米技术在生物及医药学领域的应用[J].现代化工,1999,19(7):49~51.
[11]郑俊杰,张英鸽,毛军文.纳米科技在生物医学中的应用[J].军事医学院院刊,2004,28(1):88~91.
[12]史立秋、张国顺、孙涛. AFM的纳米硬度测试与分析[J].光学精密工程,2007,15(5):725-729.
[13] Britta W.,Zhiyuan H., Xiaoping H. Molecular Portraits and 70-Gene Prognosis Signature Are Preserved Tthroughout the Metastatic Process of Breast Cancer[J]. Cancer Res,2005,65(20):9155-9158.
[14]张云艳、靳红、乔文娟. TGF—? 1促进卵巢癌细胞转移机制的研究[J].实用肿瘤学杂志,2009,23(5):417-423.
[15] Darling M.,Zauscher S.,Block A. A Thin-Layer Model for Viscoelastic, Stress-Relaxation Testing of Cells Using Atomic Force Microscopy: Do Cell Properties Re?ect Metastatic Potential?[J]. Biophysical Journal,2007,92(1):1784-1791.
[16] Roger D. Cellular Fluid Mechanics[J]. Annual Review of Fluid Mechanics,2002,34(1):211-232.
[17]刘肖珩.稳定剪切流动下黏附于血管表面的白细胞发生变形的生物力学机制[J].中国科学,2003,33(5):473-480.
[18] Q.S.Li,AFM Indentation Study of Breast Cancer Cells[J]. Biochemical and Biophysical Research Communications,2008,374(1):609-613.
[19]傅遍红,张述林,吴泽志.整合素? 2 ?1对肝癌细胞粘附与趋化行为的影响[J].重庆大学学报,2007,30(2):89-92.
[20] Guck J.,Ananthakrishnan R.,Mahmood. The Optical Stretcher: A Novel Laser Tool to Micromanipulate Cells[J]. Biophysical Journal,2001,81(2):767-784.
[21] Mahaffy R.,Park S.,Gerde E. Quantitative Analysis of the Viscoelastic Properties of Thin Regions of Fibroblasts Using Atomic Force Microscopy[J]. Biophysical Journal,2004,86(3):1777-1793.
[22] Xiaodong Li.,Bharat B. A Review of Nanoindentation Continuous Stiffness Measurement Technique and Its Applications[J]. Materials Characterization,2002,48(1):11-16.
[23]谢存毅.纳米压痕技术在材料科学中的应用[J].实验技术,2001,30(1):432-435.
[24]黎明,温诗铸.纳米压痕技术理论基础[J].机械工程学报,2003,39(3):142-145.
[25] Binning G.,Quate C. Atomic Force Microscope[J]. Phys. Rev. Lett,1986,56(1):930-933.
[26] Calabri L.,Pugno N.,Menozzi C. AFM Nanoindentation: Tip Shape and Tip Radius of Curvature Effect on the Hardness Measurement[J]. Journal of Physics,2008,20(47):223-231.
[27] Rabinovich Y.,Daosukho S.,Byer K. Direct AFM Measurements of Adhesion Forces between Calcium Oxalate Monohydrate and Kidney Epithelial Cells in the Presence of Ca2+ and Mg2+[J] Colloid Interface Sci,2008,325(2):594~601.
[28] Tatyana G. Atomic Force Microscopy Probing of Cell Elasticity[J]. Micron, 2007,38:824~833.
[29] Crick S.L.,Yin F. Assessing Micromechanical Properties of Cells with Atomic Force Microscopy: Importance of the Contact Point[J]. Mechanobiol,2007,6 (3) :199–210.
[30] Smith B.,Tolloczko., Martin.J.P. Probing the Viscoelastic Behavior of Cultured Airway Smooth Muscle Cells with Atomic Force Microscopy: Stiffening Induced by Contractile Agonist[J]. Biophys,2005,88 (4):2994–3007.
[31] Bhushan B.,Koinkar V. Nanoindentation Hardness Measurements Using Atomic Force Microscopy[J]. Am. Institute Phy,1994,64 (13):1653—1655.
[32]周兆兵、张洋、王思群.基于AFM的木质材料的纳米压痕技术[J].林产工业,2007,23(2):23-26.
[33]闫永达、尹大勇、孙涛. SPM微悬臂弹性常数校准技术[J].微纳米电子技术,2009,46(3):174-181.
[34] Yongda Y.,Shen D.,Tao S. 3D Force Components Measurement in AFM Scratching Tests[J]. Ultramicroscopy,2005,105(1—4)l 62—71.
[35] Yongda Y.,Tao S.,Shen D. Study on Effects of Tip Geometry on AFM Nanoseratching Tests[J]. Wear,2007,262(3/4):477—483.
[36] Cleveland J.,Msnne S.,Bocek D. A Nondestructive Method for Determining the Spring Constant of Cantilever for Scanning Force Microscopy[J]. Rev Sci Instrum,1993,64(2):403—405.
[37] Sader J.,Larson I.,Mulvahey P. Method for the Calibration of Atomic Force Microscope Cantilevers[J]. Rev Sci lnstrum,1995,66 (7):3789—3798.
[38] Holbery J.,Eden V.,Sarikaya M. Experimantal Determination of Scanning Probe Microscope Cantilever Spring Constants Utilizing a Nanoindentation Apparatus[J]. Rev Sci Instrum,2000,71(10):3769—3776.
[39]刘炜、蔡莉、宁金峰. Clusterin在Anip973/NVB细胞株中的表达及其意义[J].中国肺癌杂志,2008,11(5):696-699.
[40]尹梅梅、潘振伟、蔡本志.二氢杨梅素诱导人肺腺癌细胞系AGZY-83a凋亡的实验研究[J].中国药理学通报,2008,24(5):626-629.
[41] Alcaraz J.,Buscemi L.,Grabulosa M. Microrheology of Human Lung Epithelial Cells Measured by Atomic Force Microscopy[J]. Biophysical Journal,2003,84(5):2071–2079.
[42] Butt H.,Cappella B.,Kappl M. Force Measurements with the Atomic Force Microscope: Technique, Interpretation and Applications[J]. Surface Science Reports,2003,59(2):1–152.
[43] Huang H.,Kamm R.,Lee R. Cell Mechanics and Mechanotransduction: Pathways, Probes and Physiologyp[J]. American Journal of Physiology,2004,287(23):1–11.
[44] Trickey W.R.,Lee G.M.,Guilak F. Viscoelastic Properties of Chondrocytes From Normal and Osteoarthritic Human Cartilage[J]. Orthop Res,2000,18(6):891-898.
[45] Trickey W.R, Vail T.P, Guilak F. The Role of the Cytoskeleton in the Viscoelastic Properties of Human Articular Chondrocytes[J]. Orthop Res 2004,22(1):131-139.
[46] Tomkoria S.,Patel R.V.,Mao JJ. Heterogeneous Nanomechanical Properties of Superficial and Zonal Regions of Articular Cartilage of the Rabbit Proximal Radius Condyle by Atomic Force Microscopy[J]. Med Eng Phys, 2004,26(10):815-822.
[47] Bader D.L.,Ohashi T.,Knight MM. Deformation Properties of Articular Chondrocytes: A Critique of Three Separate Techniques[J]. Biorheology,2002,39(1-2):69-78.
[48] Harding J.W. , Sneddon I.N. The Elastic Stresses Produced by the Indentation of The Plane Surface of a Semi-infinite Elastic Solid by a Rigid Punch[J]. Proc Cambridge Philos Soc,1945,41:16-26.
[49] Theret D.P.,Levesque M.J.,Sato M. The Application of a Homogeneous Half-space Model in the Analysis of Endothelial Cell Micropipette Measurements[J]. Biomech Eng,1988,110(3):190-199.
[50] Darling E.,Zauscher S.,Guilak F. Viscoelastic Properties of Zonal Articular Chondrocytes Measured by Atomic Force Microscopy[J]. Osteoarthritis Cartilage,2006,14(6):571–579.
[51] Darling E.,Hu J.,Athanasiou K.A. Zonal and Topographical Differences in Articular Chondrocyte Gene Expression[J]. Orthop Res, 2004,22(6):1182-1187.
[52]杨谦,孙润广.红细胞膜弹性特性的AFM力曲线测量[J].生命科学, 2008,38(11):1013~1027.
[53] Park S.,Costa K.D.,Ateshian G.A. Microscale Frictional Response of Bovine Articular Cartilage from Atomic Force Microscopy[J]. Biomech, 2004,37(11):1679-1687.
[54] Hu K.,Radhakrishnan P.,Patel RV. Regional Structural and Viscoelastic Properties of Fibrocartilage upon Dynamic Nanoindentation of the Articular Condoyle[J]. Struct Biol,2001,136(1):46-52.
[2]袁倬斌.有机分析研究进展—单原子、单分子、单细胞的分析进展[J].分析实验室,2002,21(1):97-104.
[3] Gabriel Y.,Chwee T. Biomechanics Approaches to Studying Human Diseases[J]. Trends in Biotechnology,2007,25(3):111-118.
[4] Sergey N. Single-cell Analysis Avoids Sample Processing Bias[J]. Journal of Chromatography B,2000,741(1) :31~35.
[5] Benjamin A. Cellular Biomechanics Investigated by Atomic Force Microscopy[D]. Montreal:Thesis Submitted to McGill University for the Degree of Doctor,2004:4-6.
[6]张经济.追踪癌细胞转移之谜[J].中国医药指南,2003,05:48-49.
[7] Hanahan D.,Weinberg R. The Hallmarks of Cancer[J]. Cell,2000,100(1):57-70.
[8] Susana M.,Rafael B. Stress Relaxation Microscopy: Imaging Local Stress in Cells[J]. Journal of Biomechanics,2010,43(1):349-354.
[9]胡德红,龚萍,蔡林涛.纳米技术在癌症早期诊断和治疗中的研究与展望[J].先进技术研究通报,2009,03 (01):7~12.
[10]张汝冰,刘宏英,李凤生.纳米技术在生物及医药学领域的应用[J].现代化工,1999,19(7):49~51.
[11]郑俊杰,张英鸽,毛军文.纳米科技在生物医学中的应用[J].军事医学院院刊,2004,28(1):88~91.
[12]史立秋、张国顺、孙涛. AFM的纳米硬度测试与分析[J].光学精密工程,2007,15(5):725-729.
[13] Britta W.,Zhiyuan H., Xiaoping H. Molecular Portraits and 70-Gene Prognosis Signature Are Preserved Tthroughout the Metastatic Process of Breast Cancer[J]. Cancer Res,2005,65(20):9155-9158.
[14]张云艳、靳红、乔文娟. TGF—? 1促进卵巢癌细胞转移机制的研究[J].实用肿瘤学杂志,2009,23(5):417-423.
[15] Darling M.,Zauscher S.,Block A. A Thin-Layer Model for Viscoelastic, Stress-Relaxation Testing of Cells Using Atomic Force Microscopy: Do Cell Properties Re?ect Metastatic Potential?[J]. Biophysical Journal,2007,92(1):1784-1791.
[16] Roger D. Cellular Fluid Mechanics[J]. Annual Review of Fluid Mechanics,2002,34(1):211-232.
[17]刘肖珩.稳定剪切流动下黏附于血管表面的白细胞发生变形的生物力学机制[J].中国科学,2003,33(5):473-480.
[18] Q.S.Li,AFM Indentation Study of Breast Cancer Cells[J]. Biochemical and Biophysical Research Communications,2008,374(1):609-613.
[19]傅遍红,张述林,吴泽志.整合素? 2 ?1对肝癌细胞粘附与趋化行为的影响[J].重庆大学学报,2007,30(2):89-92.
[20] Guck J.,Ananthakrishnan R.,Mahmood. The Optical Stretcher: A Novel Laser Tool to Micromanipulate Cells[J]. Biophysical Journal,2001,81(2):767-784.
[21] Mahaffy R.,Park S.,Gerde E. Quantitative Analysis of the Viscoelastic Properties of Thin Regions of Fibroblasts Using Atomic Force Microscopy[J]. Biophysical Journal,2004,86(3):1777-1793.
[22] Xiaodong Li.,Bharat B. A Review of Nanoindentation Continuous Stiffness Measurement Technique and Its Applications[J]. Materials Characterization,2002,48(1):11-16.
[23]谢存毅.纳米压痕技术在材料科学中的应用[J].实验技术,2001,30(1):432-435.
[24]黎明,温诗铸.纳米压痕技术理论基础[J].机械工程学报,2003,39(3):142-145.
[25] Binning G.,Quate C. Atomic Force Microscope[J]. Phys. Rev. Lett,1986,56(1):930-933.
[26] Calabri L.,Pugno N.,Menozzi C. AFM Nanoindentation: Tip Shape and Tip Radius of Curvature Effect on the Hardness Measurement[J]. Journal of Physics,2008,20(47):223-231.
[27] Rabinovich Y.,Daosukho S.,Byer K. Direct AFM Measurements of Adhesion Forces between Calcium Oxalate Monohydrate and Kidney Epithelial Cells in the Presence of Ca2+ and Mg2+[J] Colloid Interface Sci,2008,325(2):594~601.
[28] Tatyana G. Atomic Force Microscopy Probing of Cell Elasticity[J]. Micron, 2007,38:824~833.
[29] Crick S.L.,Yin F. Assessing Micromechanical Properties of Cells with Atomic Force Microscopy: Importance of the Contact Point[J]. Mechanobiol,2007,6 (3) :199–210.
[30] Smith B.,Tolloczko., Martin.J.P. Probing the Viscoelastic Behavior of Cultured Airway Smooth Muscle Cells with Atomic Force Microscopy: Stiffening Induced by Contractile Agonist[J]. Biophys,2005,88 (4):2994–3007.
[31] Bhushan B.,Koinkar V. Nanoindentation Hardness Measurements Using Atomic Force Microscopy[J]. Am. Institute Phy,1994,64 (13):1653—1655.
[32]周兆兵、张洋、王思群.基于AFM的木质材料的纳米压痕技术[J].林产工业,2007,23(2):23-26.
[33]闫永达、尹大勇、孙涛. SPM微悬臂弹性常数校准技术[J].微纳米电子技术,2009,46(3):174-181.
[34] Yongda Y.,Shen D.,Tao S. 3D Force Components Measurement in AFM Scratching Tests[J]. Ultramicroscopy,2005,105(1—4)l 62—71.
[35] Yongda Y.,Tao S.,Shen D. Study on Effects of Tip Geometry on AFM Nanoseratching Tests[J]. Wear,2007,262(3/4):477—483.
[36] Cleveland J.,Msnne S.,Bocek D. A Nondestructive Method for Determining the Spring Constant of Cantilever for Scanning Force Microscopy[J]. Rev Sci Instrum,1993,64(2):403—405.
[37] Sader J.,Larson I.,Mulvahey P. Method for the Calibration of Atomic Force Microscope Cantilevers[J]. Rev Sci lnstrum,1995,66 (7):3789—3798.
[38] Holbery J.,Eden V.,Sarikaya M. Experimantal Determination of Scanning Probe Microscope Cantilever Spring Constants Utilizing a Nanoindentation Apparatus[J]. Rev Sci Instrum,2000,71(10):3769—3776.
[39]刘炜、蔡莉、宁金峰. Clusterin在Anip973/NVB细胞株中的表达及其意义[J].中国肺癌杂志,2008,11(5):696-699.
[40]尹梅梅、潘振伟、蔡本志.二氢杨梅素诱导人肺腺癌细胞系AGZY-83a凋亡的实验研究[J].中国药理学通报,2008,24(5):626-629.
[41] Alcaraz J.,Buscemi L.,Grabulosa M. Microrheology of Human Lung Epithelial Cells Measured by Atomic Force Microscopy[J]. Biophysical Journal,2003,84(5):2071–2079.
[42] Butt H.,Cappella B.,Kappl M. Force Measurements with the Atomic Force Microscope: Technique, Interpretation and Applications[J]. Surface Science Reports,2003,59(2):1–152.
[43] Huang H.,Kamm R.,Lee R. Cell Mechanics and Mechanotransduction: Pathways, Probes and Physiologyp[J]. American Journal of Physiology,2004,287(23):1–11.
[44] Trickey W.R.,Lee G.M.,Guilak F. Viscoelastic Properties of Chondrocytes From Normal and Osteoarthritic Human Cartilage[J]. Orthop Res,2000,18(6):891-898.
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