【作者】 金花；

【导师】 蔡继业；

【作者基本信息】 暨南大学 ， 无机化学， 2009， 硕士

【摘要】 本文第一部分基于原子力显微术,结合平板菌落计数法,系统探究了血卟啉单甲醚(HMME)对革兰氏阳性菌一金黄色葡萄球菌(S.aureus)和革兰氏阴性菌—大肠杆菌(E.coli)形态结构的影响及其抗菌作用机理。并在此基础上对卟啉类光敏剂的光动力杀菌机理进行了验证和补充,即HMME对细菌的杀伤作用与HMME的浓度有关,也与细菌的种类有关。同浓度下,光照条件下比无光照时对S.aureus的杀灭效果显著。同等条件下,无论光照还是无光照,HMME对E.coli都没有明显的杀伤作用。HMME在光照作用下对S.aureus表面结构的破坏较E.coli严重,AFM图像显示无论是G~+还是G~-在HMME的光动力作用下,都是细菌细胞表面受到严重损伤,未发现内容物泄漏,说明膜损伤是PDT致细菌死亡的一个主要机制。AFM从形貌学上证明了HMME的APDT机理是选择性的攻击细菌细胞的膜性结构。本文的第二部分是关于AFM对小鼠间充质干细胞表面CD44抗原分子的单分子识别。用AFM力谱测量软件,探测细胞与裸针尖之间的非特异性粘附力,同时还通过杨氏模量计算方法,获得细胞表面的粘弹性等参数;发展了原子力“针尖化学”技术,在原子力针尖上修饰CD44单克隆抗体,特异性识别细胞表面CD44抗原分子,实现了定量测量干细胞表面CD44抗原和抗体分子之间的作用力。探讨和研究了原子力显微镜对细胞表面大分子识别的可行性和意义。用原子力显微镜研究生物大分子在细胞膜上的分布、定位及运动,将在分子识别、信号转导以及细胞活化等领域大显身手。本文第三部分采用原子力显微镜(AFM)对10例2型糖尿病患者和8例健康志愿者的红细胞膜的机械性质和形貌进行了纳米力学测量。方法:将AFM悬臂定位在细胞顶端,在细胞上500nm~2的范围内随机选取100个以上的点,扫描范围设置为0nm,采用AFM接触成像模式中的力调节模式测得,记录的力曲线用EXCEL2003统计比较。结论:2型糖尿病患者的红细胞比健康人的硬度和粘附力都明显增大,细胞的高度(Mh)、细胞最高点与最低点的差值(Rp-v)显著降低,而直径增大。AFM为我们从纳米力学方面表征红细胞的病理特征提供了有力工具。更多还原

【Abstract】 Because of the nano-meter/subnano-meter resolution and simple sample preparation procedure,atomic force microscopy(AFM) has been proved a powerful tool to obtain topography and biomechanical properties of various materials.In author’s experiments, AFM was used as an important means to detect the photoactivation effects of hematoporphvrin monomethyl on Gram-positive bacteria -Staphylococcus aureus and Gram-negative bacteria -Escherichia coli and the CD44 single-molecule recognition events on mesenchymal stem cell surface.In the first part,the photodynamic inactivation of Gram-positive bacteria-Staphylococcus aureus and Gram-negative bacteria- Escherichia coli by hematoporyrin monomethyl ether was investigated by the reduction of colony unit and AFM.Results indicated that 90%of Staphylococcus aureus was photoinactivated by illumination with 30 min visible light(power density 200 mW/cm~2) in the presence of 50μg/mL HMME. The antiactivation efficiency to Staphylococcus aureus with light irradiation was much obvious than that in dark at the same concentration of HMME,but there was not noticeable damage to E.coli with illumination or in dark.AFM ultrastructure images showed that the cell surface of photodynamic inactivated bacteria was all damaged seriously but there’s no cell contents leaked.So we concluded that the attacked sites to bacteria cells by hematoporyrin monomethyl ether were bacteria membrane structure. And atomic force microscopy provides us a visual technique to study the mechanism of bacteria reacted with photosensitizers.In the second part,Functionalized AFM was used to study the single-molecule recognition events on the surface of the rat mesenchymal stem cells(MSCs).Molecular recognition plays an important role in biological systems and is observed between receptor-ligand,antigen-antibody,DNA-protein,sugar-lectin,RNA-ribosome,etc.To assess the specificity of recognition events,control experiments have been performed by recording force-distance curves using modified and unmodified AFM tips,respectively. Under these conditions,thousands of force-curves have been acquired over different sites of the sample and the unbinding force of the observed events has been measured.In the results,we obtained the CD44 molecule distribution on the surface of MSCs which indicated the CD44 molecule distribution was not homogeneous.Simultaneously,from force-curve analysis we estimated the interaction force is around 250 pN.In the present, AFM is the only force-measuring technique that can map the nanoscale lateral distributon of single molecular recognition sites on biosurfaces.In the third part,AFM was used to measure the morphology and nanomechanical properties(elasticity and adhesion) of erythrocyte taken from type 2 mellitus patients and healthy people seperately.The results showed that stiffness and adhesion of diabetes patients’ erythrocyte were higher than healthy people’s.In morphology,the patient’ erythrocyte was much lower in cell median height(Mh)and cell peak to valley(Rp-v), but bigger higher in diameter than those of the erythrocyte of healthy people.AFM provides us more detailed data for better understanding the pathologic characteristics of erythrocyte and has been proved to be a powerful tool in disease detecting and diagnosis.更多还原