扫描探针显微术加量子点示踪研究特异性T细胞的抗原识别和活化

项目名称： 扫描探针显微术加量子点示踪研究特异性T细胞的抗原识别和活化

项目编号： No.30872404

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 蔡继业

作者单位： 暨南大学

项目金额： 30万元

中文摘要： 扫描探针显微镜能够探测单个大分子在细胞表面和细胞内的运动而不损害细胞。量子点标记有荧光寿命长、激发谱宽、发射谱窄的优点。我们利用近场光学显微镜实现了突破衍射极限的50nm高分辨；采用量子点标记细胞分子并利用电子调谐的方法达到同时原位探测两种或三种细胞表面分子的分布；利用针尖化学方法，将修饰有特异性抗体分子的原子力显微镜针尖原位测量细胞表面单个抗原-抗体分子的相互作用力。利用上述高分辨、高精度的实验方法，研究了各种T细胞（如CD4+、CD8+、Jurkat T细胞等）表面抗原分子（如CD8、CD69、CD59、GM1脂筏等）在不同的药物、病原体刺激前后在细胞表面分布的变化、抗原-抗体相互作用力的变化和生物物理性质的变化。如首次证明了T细胞活化过程中CD69分子在细胞表面的纳米机械性质及分布特征；青蒿琥酯(ART)能抑制Jurkat T细胞的分化、改变细胞的机械性质；发现了PDB刺激的CD8+T细胞表面粘附力增加；低温诱导脂筏标记GM1与CD59形成了共定位的纳米簇和异质性的功能生物膜等。这将为抗病菌、病毒和抗肿瘤疫苗的制备提供重要实验和理论依据。

中文关键词： 扫描探针显微镜； 量子点 ；T细胞 ；单分子探测

英文摘要： Single molecule on cell surface and inside cell can be detected by scanning probe microscopy without destroying the cell itself. Quantum dots marked molecules have long fluorescent life time, wider excitation bands, and narrow emission bands. NSOM was used to reach 50nm resolution; tip chemistry was used to detect in situ antibody-antigen interaction force and different QD marked molecules were recognized by electric tuned NSOM. These high resolution and high precision method were successfully used to detect single molecule distribution (including CD8, CD69, CD59, GM1 lipid rafts etc) on different T cell(including CD4+, CD8+, Jurkat T cells etc) under different drug and pathogen stimulation. we are the first time to demonstrate nano-biology of CD69 expression during T cell activation; ART-induced biophysical and biochemical alterations of Jurkat cell membrane; the adhesion force of membrane was increased after CD8+ T cells were stimulated with PDB; cold induces micro- and nano-scale reorganization of lipid raft markers(GM1) at mounds of T-cell membrane fluctuations, etc. This study provide important experiment and theory results to develop antimicrobial vaccine.

英文关键词： scanning probe microscopy; Quantum dots; T cell; single molecule detection