摘要
纳米生物技术是纳米技术与生命科学的交叉,是在纳米尺度上形成的有关生命科学的纳米新技术和新方法,在分子、亚细胞和细胞水平上研究生物分子和细胞的行为和相互作用机制具有独特的优势。本论文基于AFM和NSOM,结合量子点标记及激光共聚焦等技术,以人外周血T淋巴细胞(包括特异性和非特异性)为研究对象,在纳米尺度研究了活化前后以及不同活化阶段T细胞生物物理特性的变化,即细胞形态结构、膜表面纳米结构、膜孔变化、膜表面摩擦力分布、膜表面粘附特性、抗原-抗体间特异性相互作用力、膜表面受体分子分布模式等。并以红细胞为模型初步探讨了AFM的应用价值与可行性。主要获得了以下具有创新性的结果:
1)完成了静息和处于不同活化阶段T淋巴细胞的纳米结构的定性、定量分析。经PDB加ION刺激活化后,CD_3~+T细胞的直径、体积、膜表面纳米颗粒的平均高度均增大,但膜表面平均粗糙度无显著性差异:CD_4~+及CD_8~+T细胞的各项物理参数均增大:在纳米尺度,CD_4~+和CD_8~+T细胞膜表面的摩擦力分布不均匀,揭示了膜表面化学成分及物理特性的非均质性;
2)发现活化前后及不同活化阶段,T细胞表面力学特性发生显著变化。在气相中测量发现,静息CD_3~+T的表面粘附力为1025±799.84 pN,随着活化时间的延长,细胞膜表面粘附力变小,其中刺激24小时的膜表面粘附力最小,为243.93±167.51 pN;对CD_4~+T细胞而言,活化24小时的膜表面粘附力最大(827.6±271.8 pN),约为静息组及活化48和72小时组膜表面粘附力的一倍;同样,活化后CD_8~+T细胞表面的粘附力(300～800 pN)大于静息组细胞表面的粘附力(100～700 pN),均符合正态分布。但在液相中的测量发现,裸探针与CD_4~+及CD_8~+T细胞(包括静息及活化)膜表面无明显的非特异性粘附力及非线性作用力。
3)液相中对活化前后CD_4~+T细胞原位测量发现,CD_4抗原-抗体间作用力(200-1200pN)大于和CD_(69)抗原-抗体间作用力(30-210 pN);对CD_8~+T细胞的测量显示,CD_8抗原-抗体间的作用力(918±430.6 pN)稍小于CD_(69)抗原-抗体分子的特异性作用力(1097.8±675.2 pN)。特异性作用的力曲线均表现出明显的非线性作用力特性;
4)三维重构图、二维灰阶模式图以及激光共聚焦荧光成像均表明,CD_4分子及活化标志分子CD_(69)在CD_4~+T细胞膜表面呈不均匀分布,主要以纳米簇或微结构域的形式存在。NSOM荧光图(实验最高分辨率为92 nm)同样显示CD_4和CD_(69)分子在膜表面呈不均匀状态,超过75%的CD_4分子在细胞膜上呈聚集状态,聚集成微/纳结构域,形成脂筏或微功能结构域,而荧光团的面积、直径及荧光强度的计算和统计也符合这一结果;对CD_8~+T细胞而言,膜表面受体分子分布模式的三维重构及灰阶模式图显示CD_8分子和CD_(69)分子在膜表面亦呈不均匀分布,聚集成微/纳结构域;
5)初步探讨了AFM力谱应用于医学、法医学检验的价值与可行性。以红细胞为模型,测量了在室温、室外环境及低温等三个条件下,玻璃和云母基底上细胞形态学及膜表面粘附力随时间变化的关系。在室温条件下,云母基底上红细胞体积的减小幅度较玻璃基底上细胞明显;在室外条件下,玻璃基底上红细胞体积先增大,再减小,而云母基底上细胞体积则先增大,再显著减小,而后出现平台。对于粘附力而言,在室温和室外条件下,两种基底上红细胞的粘附力均先增大再减小,但是曲线最高点出现的时间点不同。在低温条件即4℃下,细胞体积随时间延长而急剧减小,在第5天时,细胞完全塌陷;细胞表面粘附力也随时间的延长而减小。
综上所述,本文利用纳米生物技术第一次在纳米尺度对T细胞活化前后生物物理特性的变化进行了较为深入详细的研究分析,获得了大量有价值的实验数据与信息,为进一步研究T细胞介导的免疫反应与识别提供了新的研究视角和思路。对红细胞在不同条件下随时间变化相关性的探讨揭示了AFM应用于医学或法医学检验甚至临床疾病诊断的价值与潜力。由于以AFM和NSOM为代表的扫描探针显微术具有纳米结构和纳米力学特性的可获得性,因此它们在纳米生物学领域的研究中将扮演不可或缺的角色。而纳米生物技术作为纳米科技研究的前沿交叉领域,必将引起人们的更大重视和关注,因此纳米生物技术领域是一个大有可为的领域,将为解决生命科学中的一些重大问题做出更积极的贡献。
Nanobiotechnology,as the cross-discipline of nanotechnology and life science,is a novel technology and approach in nanoscale life science study;it can provide many insightful views into biophysical behaviors of biomolecules and cells including the intramolecular force, intermolecular interaction,and molecule-molecule and cell-cell interaction mechanism.In this work,atomic force microscopy(AFM),near-field scanning optical microscopy,laser scanning confocal microscopy and quantum dot labeling were used to investigate the biophysical properties(the parameters including morphology,membrane nanostructures,membrane pore, friction distribution,adhesion properties,specific interaction of antigen-antibody,distribution mode of membrane receptors and so on) of T lymphocytes stimulated by phorbol dibutyrate (PDB) plus ionomycin(ION) at nanoscale.In addition,the biophysical variation of erythrocytes with the time lapse was analyzed utilizing AFM.According to the experiments,we obtained many insightful results shown as the followings:
1) The nanostructures of resting and activated T lymphocytes were firstly evaluated.The results indicate that cell diameter,cell volume and the mean height of membrane surface particles of CD3+ T cells increased due to PDB plus ION stimulation;as a contrast,all biophysical parameters of CD4+ and CD8+ T cells increased after stimulation.The lateral force measurements indicate the heterogeneous distribution of friction on CD4+ and CD8~+ T cell membrane,revealing the chemical and biophysical heterogeneity;
2) Nano-mechanical properties of resting T cells evidently differ from activated T cells.The results obtained in air indicate that the surface adhesion force of resting CD3~+ T cells is 1025±799.84 pN,which decreased with the lapse of activation time,and that measured at 24 hours stimulation time appears to be the lowest(243.93±167.51 pN);whereas,the adhesion force of CD4~+ T cells measured at 24 hours stimulation time is the largest(827.6±271.8 pN) among three experimental groups,24,48 and 72 hours group,and force curves of CD4~+ T cells show unobvious snap-in force;as for CD8~+ T cells,the adhesion force of activated cell distributes between 300 pN and 800 pN,which is also larger than that of resting cells,100～700 pN.When the measurements were conducted in liquid,the force curves indicate that adhesion force and nonlinear interaction between bare tip and CD4~+,CD8~+ T cells are not obvious.
3) As for specific interaction of antigen-antibody of CD4+ T cells measured in situ,the results exhibit that the specific interaction force of CD4 antigen-antibody is 200-1200 pN,which is larger than that of CD69 antigen-antibody(30-210 pN);as for CD8~+ T cells,however,the specific interaction force of CD8 antigen-antibody is 918±430.6 pN,which is slightly smaller than that of CD69 antigen-antibody(1097.8±675.2 pN);moreover,the specific force curves reveal the evident nonlinear interaction between tip and cell membrane;
4) Investigation of distribution mode of membrane receptors.3-D reconstruction map, grayscale map and imaging results of laser scanning confocal microscopy altogether demonstrate that both CD4 receptors and activation marker CD69 receptors non-uniformly distribute on CD4~ T cell membrane,concentrating into nano-clusters and/or micro-domains.Furthermore, near-field fluorescent images,whose highest resolution is 92 nm(FWHM),also reveal the inhomogeneous distribution of CD4 and CD69 receptors on cell membrane;the statistics further indicates that more than 75%CD4 molecules on cell membrane concentrates into nano-/microstructural domains,which is supported by the analysis of area,diameter and fluorescent intensity of near-field fluorophores.As for CD8~+ T cells,the 3-D reconstruction map and grayscale map, which imply the distribution mode/pattern of membrane receptors,also illustrate that CD8 and CD69 receptors inhomogeneously distributing on cell membrane and forming nano-/microstructural domains:
5) Atomic force microscopy(AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains,potentially providing useful information for forensic investigation.Here,the time-dependent,morphological changes of red blood cells(RBC) under three conditions(including controlled,room-temperature condition,uncontrolled, outdoor-environmental condition,and controlled,low-temperature condition) were observed by AFM,as well as the cellular viscoelasticity via force-vs-distance curve measurements.With time lapse,the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition.Under the controlled low-temperature condition,however,the changes in cell volume occurred mainly due to the collapse of RBCs,and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC.
In the present work,the variations of biophysical properties of Tlymphocytes in the context of activation in vitro were evaluated at nanoscale for the first time,and the obtained experimental data and information offer us new insightful views for further understanding/elucidating immune response/recognition correlated biophysical behaviors of specific/non-specific T cells.On the other hand,the AFM detections on the time-,environment(temperature/humidity)- dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in medicine/forensic medicine exmination.AFM and NSOM,as two important members of scanning probe microscopy family,play crucial roles in nano-biology study due to their acquiring capabilities of nanostructures and nano-mechanical properties.As the frontier field of nanotechnology study,nanobiotechnology has aroused scientist's extensive concerns and will provide invaluable implications and insights into biophysical behaviors/processes of biomolecules and cells at nanoscale.
引文
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