【作者】 胡明铅；

【导师】 蔡继业；

【作者基本信息】 暨南大学 ， 应用化学， 2009， 硕士

【摘要】 本学位论文主要分为两大部分:（1）应用原子力显微镜（Atomic ForceMicroscope,AFM）的高分辨率和力谱特性,探测淋巴细胞在不同刺激作用下的形态变化及其粘附力和杨氏模量的变化;（2）应用原子力显微镜和（ScanningNear-field Optical Microscope,SNOM）对细胞表面受体识别进行可视化研究。本文第一部分基于原子力显微术的超高分辨率和力谱特性,应用赫兹模型（Hertz model）探测淋巴细胞的粘附力（Adhesion force）和杨氏模量（Young’smodulus）的变化。比较了静息（Resting）、脂多糖（LPS）和伴刀豆蛋白A（ConA）活化的淋巴细胞的形态结构、超微结构和粘附力特性。从AFM图像可知,经LPS或ConA刺激活化后的淋巴细胞比静息状态的淋巴细胞有所增大,并且表面分布着大小不一的颗粒状聚合物。利用AFM高空间分辨的力位移曲线测量系统,发现经LPS或ConA刺激活化后淋巴细胞的粘附力是静息状态淋巴细胞的2～3倍。利用高灵敏度的力谱测量系统,分析比较了静息（Resting）,活化（Activated）和凋亡（Apoptotic）淋巴细胞的弹性模量,结果表明活化的淋巴细胞的硬度（～20kPa）是其他静息和凋亡淋巴细胞的（5～11 kPa）2～3倍。此结果有助于我们认识细胞生长与分化各个阶段的生物力学特性。本文第二部分基于原子力显微术和针尖修饰技术,结合扫描近场光学显微术和荧光半导体量子点（Quantum dots,QDs）标记技术、倒置荧光显微镜、共聚焦显微镜与流式细胞术等进行与生物分子的结构与功能相关的免疫识别研究。本实验发展了一种方法即采用抗体功能化修饰的AFM探针,在AFM的针尖上修饰生物素化的抗体,探测细胞膜表面特异性受体分子的相互作用力。我们发现SNOM与QDs结合成为一种很好的纳米荧光成像探测系统,能对细胞表面的受体分子进行纳米尺度的空间分布成像。本文采用AFM和SNOM对人外周血CD₄⁺T细胞膜表面的CD₄和CD₆₉分子的分布情况及力谱特性进行对比研究及分析比较,主要研究结果如下:（1）来用功能化修饰的AFM针尖,测量了CD₄抗原-抗体之间的相互作用力,发现CD₄抗原-抗体之间存在明显的相互作用,其特异性作用力大约是非特异性力的3倍,其粘附力并非随即分布,而是在细胞表面聚集成纳米簇;（2）随着活化时间的增加,CD₄抗原-抗体之间存在的键合越来越多;（3）利用同样的针尖修饰方法,定量测量了CD₆₉抗原-抗体之间的相互作用力,大小为276±71 pN:（4）SNOM结合QDs探针,探测了CD₆₉分子在细胞膜表面的分布情况,发现CD₆₉分子在活化的CD₄⁺T细胞表面呈80-200nm大小的团簇不均匀分布。更多还原

【Abstract】 This dissertation includes two parts:（1） Using the high-resolution and force spectrum properties of the atomic force microscopy（AFM）,detect the changes of the morphology,adhesion force and Young’s modulus properties of the lymphocyte by different stimulation;（2） Visualization of the cell surface receptors recognition was performed by AFM and scanning near-field optical microscope（SNOM）.In the first part,based on the high-resolution and force spectrum properties of the AFM,Hertz model was used to detect lymphocyte adhesion force and Young’s modulus.The morphology,ultrastructure and adhesion force properties of the resting, LPS or ConA activated human periphery lymphocyte were investigated.The AFM images revealed that the surface of the lymphocyte treated with LPS or ConA were rougher than that of resting lymphocyte,and coated with an outer layer of extracellular polymers.High-spatially resolved force-distance curves indicated that the adhesion force values of lymphocyte activated by LPS or ConA were approximately two to three times stronger than that of resting lymphocyte.Using high-sensitivity force-distance curves,the Young’s modulus of the resting,activated and apoptotic lymphocyte were detected.We found that the value of the activated lymphocyte is about two to three times stiffer（Young’s modulus of～20 kPa） than those of the two other morphotypes（5～11 kPa）.These results can improve our understanding the mechanical properties of cells during growth and differentiation.The second part of this paper,based on AFM and tip modification technology, combined with SNOM,fluorescent quantum dots（QDs） mark,fluorescence microscope,confocal microscope and flow cytometry,the structure and function or the immune recognition of the biological molecules were being studied.A method involving the antibody functionalized modified AFM tip to detect specific cell surface receptor molecule interaction.To determine the nanoscale distribution of molecules on cell-membrane,Ab-functionalized AFM tip was tested for its atomic force binding to molecules potentially expressed on the cell membrane.The AFM tip was first treated with the strepavidin.The strepavidin-labeled AFM tip was then attached to biotinylated Ab.We have found that SNOM/QD-based nanoscale fluorescence imaging is a powerful system in which to visualize nano-spatial distribution and organization of cell-surface immune molecules.The distributive patterns and force properties of CD₄ and CD₆₉ molecules on human Peripheral Blood CD₄⁺ T cell surface were compared by AFM and SNOM.The main results are the following:（1） Using a functionalized AFM tip,the strength of the specific binding force of the CD₄ antigen-antibody interaction was found to be approximately three times that of the unspecific force.The adhesion forces were not randomly distributed over the surface of a single activated CD₄⁺ T cell indicated that the CD₄ molecules concentrated into nanodomains;（2） Multiple bonds involved in the CD₄ antigen-antibody interaction were measured at different activation times;（3） Using the same method,quantitative measurement of the CD₆₉ antigen-antibody interaction,about 276±71 pN;（4） SNOM/QD-based nanoscale imaging showed that CD₆₉ were non-uniformly distributed as 80-200 nm nanoclusters on cell-membrane of activated CD₄⁺ T cell.更多还原