基于原子力显微术的光动力杀菌机理和干细胞表面单分子识别的研究
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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。
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.
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。
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.
引文
[1]Y.De Wilde and P.-A.Lemoine Review of NSOM Microscopy for Materials,AIP Conf.Proc.2007,931- 943.
[2]Muller DJ.AFM:a nanotool in membrane biology,Biochemistry,2008,47(31):7986-7998.
[3]Pierre Parot,Yves F.Dufre ^ne,Peter Hinterdorfer,Christian Le Grimellec,Daniel Navajas,Jean-Luc Pellequer and Simon Scheuring.Past,present and future of atomic force microscopy in life sciences and medicine.J.Mol.Recognit.2007,20:418-431.
[4]F.J.Rubio-Sierra,W.M.Heckl,R.W.Stark.Nanomanipulation by Atomic Force Microscopy,Advanced Engineering Materials,2005,7:193 - 196.
[5]C.Baur et al.,"Nanoparticle manipulation by mechanical pushing:underlying phenomena and real-time monitoring",Nanotechnology,1998,9(4):360-364.
[6]R.M.Taylor et al.,"The nanomanipulator:a virtual reality interface for a scanning tunneling microscope",Proc.ACM SIGGRAPH,Anaheim,CA,1993,8:127-134.
[7]G.Binning,C.F.Quate,Ch.Gerber.Atomic force microscope.Phys.Rev.Lett.1986,56(9):930.
[8]BALDWIN,P.M,FRAZIER,R.A,ADLER,J,GLASBEY,T.O,KEANE,M.P,ROBERTS,C.J,TENDLER,S.J.B,DAVIES,M.C,MELIA,C.D.Surface imaging of thermally sensitive particulate and fibrous materials with the atomic force microscope:a novel sample preparation method.Journal of Microscopy.1996,184(2):75-80.
[1]Ana P.Castano,Pawel Mroz,Michael R.Harnblin.Photodynarnic therapy and anti-tumour Immunity,Nat Rev Cancer.2006,6:535-545.
[2]唐蕾,诸葛健.天然光敏剂的光动力杀伤作用[J].中国抗生素杂志,2004,29(11):703-706.
[3]Yoshimura M.,Namura S.,Akamatsu H.and Horio T..Antimicrobial effects of phototherapy and photochemotherapy in vivo and in vitro[J].British Dermatology,1996,135:528-532.
[4]M.Wainwright,Photoantimicrobials - a PACT against resistance and infection,Drugs of the Future,2004,29:85-93.
[5]Cecilia Bombelli,Federico Bordi,Stefania Ferro,et al.New Cationic Liposomes as Vehicles of m-Tetrahydroxyphenylchlorin in Photodynamic Therapy of Infectious Diseases Mol.Pharmaceutics,2008,5(4),672-679
[6]Tim Maisch.Anti-microbial photodynamic therapy:,useful in the future? Lasers Med Sci,2007,22:83-91.
[7]FOOTE C S.Type Ⅰ and Type Ⅱ Mechanisms of Photodynamic Action.In Light Activeted Pesticides[M].Wastington:American Chemical Society,1987,22228.
[8]Nitzan Y.,Gutterman M.and Malik Z..Inactivation of gram negative bacteria by photosensitized porphyrins[J].Photochemistry and Photobiology.1992,55:89 -96.
[9]Saeki Y.,Ito Y.and Shibata M.l..Antimicrobial action of green tea extract,flavono flavor and copper chlorophyll against oral bacteria[J].Bull Tokyo DentColl,1993,34(1):33-37.
[10]North J.,Neyndorff H.and Levy J.G..Photosensitizers as virucidal agents[J].Journal of Photochemistry and Photobiology.B:Bioogyl,1993,17(2):99-108.
[11]Malik Z.,Ladan H.and Niton Y..The bactericidal activity of a deuteroporphyrin hemin mixture on grampostive bacteria.A microbiological and spectroscopic study[J].Journal of Photochemistry and photobiology,1990,B(6):419 - 430.
[12]陈文晖,余建鑫,姚建忠,沈卫镝,刘建飞,许德余.光动力治癌药血啉甲醚的药代动力学研究[J].中国激光医学杂志,2000,9(2):106-108.
[13]瞿燕,韩光范.光动力学疗法治癌光敏剂的研究进展[J].中国激光医学杂志,2006,15(1):48-55.
[14]戴维德,王雷,刘凡光,顾瑛,李晓松,丁新民,曾晶.应用激光扫描共聚焦显微成像术研究光敏剂亚细胞定位[J].中国激光医学杂志,2004,13(1):12-17.
[15]顾瑛,李峻亨,王开,江亿平,梁洁,朱建国.血啉甲醚用于光动力疗法治疗鲜红斑痣的初步临床研究[J].中国激光医学杂志,1996,5(4):201-204.
[16]郑蔚,谢树森,陆祖康.用激光荧光法诊断和定位早期肺癌[J].激光与光电子学进展,1998,(6):29-40.
[17]王伟,李辉,李家泽,尚立群,刘小兵.肺癌组织血卟啉单甲醚激光诱发药物荧光与激光诱发自体荧光的光谱区别[J].中国激光医学杂志2003,12(4):243-246.
[18]胡韶山,王齐,岳武,杨宝峰.光动力辅助显微手术治疗脑胶质瘤[J].中华神经外科杂志,2004,20(1):30-32.
[19]Xinmin Ding,,Qinzhi Xu,Fanguang Liu,Pingkun Zhou,Ying Gu,Jing Zeng,Jing An,Weide Dai,Xiaosong Li.Hematoporphyrin monomethyl ether photodynamic damage on HeLa cells by means of reactive oxygen species production and cytosolic free calcium concentration elevation,Cancer Letters,2004,216:43-54.
[20]武汉大学生物系微生物教学研究小组.微生物[M].北京科学出版社.1987.61-64.
[21]张文治.新编食品微生物学[M].中国轻工业出版社1995,5:10-23.
[22]牛天贵.张宝芹.食品微生物检验[M].中国计量出版社,2003,2:252-312.
[23]周德庆.微生物学实验教程[M].北京:高等教育出版社,2006,109-113
[24]Malik,Z.,Ladan,H.and Nitzan,Y..Photodynamic inactivation of Gram negative bacteria:problems and possible solutions[J].Photochemistry and Photobiology,1990,5(B):811-816.
[25]郑瑜谦,闫福华,陈锦灿,赵欣,李永东,袁彩,石晓莉,黄明东.一种新型光敏剂对牙周致病菌的体外抗菌研究.口腔医学研究,2006,22(4):368-371.
[26]Taylor PW,Stapleton PD,Luzio P.New ways to treat bacterial infections.Drug Discov Today,2002,7:1086-1091.
[27]Nitzan Y,Guttennan M,Malik Z,et al.Inactivation of gram-negative bacteria by photosensitized porphyrins.Photochem Photobiol,1992,55:89-96.
[28]Maisch T,Szeimies RM,Jori G,et al.Antibacterial photodynamic therapy in dermatology.Photochem Photobiol Sci,2004,3:907-917.
[29]Menezes S,Capella MA,Caldas LR.Photodynamic action of methylene blue:repair and mutation in Escherichia coli.J Photochem Photobiol B.1990,5:505-517.
[30]J.M.Bliss,C.E.Bigelow,T.H.Foster and C.G.Haidaris,Susceptibility of Candida species to photodynamic effects of Photofrin,Antimicrob Agent Chemother,2004,48:2000-2006.
[1]陈宜张,林其谁主编.生命科学中的单分子行为及细胞内实时检测[M].北京:科学出版社,2005,1-11.
[2]Pfister G,Stroh CM,Perschinka H,et al.Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.J Cell Sci,2005,118(8):1587-94.
[3]Puntheeranurak T,Wildling L,Gruber HJ,et al.Ligands on the string:single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells.J Cell Sci,2006,119(14):2960-2967.
[4]Kim H,Arakawa H,Hatae N,et al.Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM.Ultramicroscopy,2006,106(8-9):652-662.
[5]Madl J,Rhode S,Stangl H,et al.A combined optical and atomic force microscope for live cell investigations.Ultramicroscopy,2006,106(8-9):645-651.
[6]Hong Wang,Ingrid Tessmer,Deborah L.Croteau,Dorothy A.Erie,and Bennett Van Houten.Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot,Nano Lett,2008,8(6):1631-1637.
[7]Peter Scho "n,,Martin Go " rlich,Michiel J.J.Coenen,Hans A.Heus,and Sylvia Speller.Nonspecific Protein Adsorption at the Single Molecule Level Studied by Atomic Force Microscopy,Langmuir,2007,23(20):9921-9923.
[8]White,S.H.,and Wimley,W.C.Membrane protein folding and stability:Physical principles.Annu.ReV.Biophys.Biomol.Struct.1999,28:319-365.
[9]Sapra,K.T.,Park,P.S.,Palczewski,K.,and Muller,D.J.Mechanical properties of bovine rhodopsin and bacteriorhodopsin:Possible roles in folding and function.Langmuir,2008,24:1330-1337.
[10]Scheuring,S.,Seguin,J.,Marco,S.,Levy,D.,Robert,B.,and Rigaud,J.L.Nanodissection and high-resolution imaging of the Rhodopseudomonas Viridis photosynthetic core complex in native membranes by AFM.Proc.Natl.Acad.Sci. U.S.A.2003,100:1690-1693.
[11]Alattia,J.R.,Shaw,J.E.,Yip,C.M,and Prive,G G.Molecular imaging of membrane interfaces reveals mode of P-glucosidase activation by saposin C.Proc.Natl.Acad.Sci.U.S.A.2008,104:17394-17399.
[12]Monia Taranta,Anna Rita Bizzar and Salvatore Cannistraro.Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy,J.Mol.Recognit.2008,21:63-70.
[13]Berquand A,Xia N,Castner DG,Clare BH,Abbott NL,Dupres V,Adriaensen Y,Dufrene YF.Antigen bindingforces of single antilyso-zyme Fv fragments explored by atomic force microscopy.Langmuir,2005,21:5517-5523.
[14]Schwesinger F,Ros R,Strunz T,Anselmetti D,Guntherodt H-J,Honegger A,Jermutus L,Tiefenauer L,Pluckthun A.Unbinding forces of single antibody-antigen complexes correlatewith their thermal dissociation rates.Proc.Natl.Acad.Sci.U S A,2000,97:9972-9977.
[15]Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell,2006,126 (4):663-76.
[16]Barrilleaux B,Phinney DG,Prockop DJ,O'Connor KC."Review:ex vivo engineering of living tissues with adult stem cells".Tissue Eng,2006,12 (11):3007-3019.
[17]Beckmann J,Scheitza S,Wernet P,Fischer JC,Giebel B.“Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment:identification of asymmetrically segregating proteins”.Blood,2007,109 (12):5494-5501.
[18]Adewumi O,Aflatoonian B,Ahrlund-Richter L,et al.“Characterization of human embryonic stem cell lines by the International Stem Cell Initiative”.Nat.Biotechnol,2007,25 (7):803-816.
[19]Kazuhiro Sudo,Megumi Kanno,Kenichi Miharada,Saeri Ogawa,Takashi Hiroyama,Kaoru Saijo,Yukio Nakamura.Mesenchymal Progenitors Able to Differentiate into Osteogenic,Chondrogenic,and/or Adipogenic Cells In Vitro Are Present in Most Primary Fibroblast-Like Cell Populations.Stem Cells,2007,25(7):1610 -1617.
[20]Bruder SP,Fink D J,Caplan AI.Mesenchymal stem cells in bone development,bone repair,and skeletal regeneration therapy.J Cell Biochem.1994,56(3):283-294.
[21]Bielby R,Jones E,McGonagle D.The role of mesenchymal stem cells in maintenance and repair of bone.Injury,2007,38:S26-32.
[22]Chotima Boettcher.Gene-modified bone marrow-derived stem cells:an attractive gene delivery system in inherited retinal disorders,Asian Biomedicine 2007,1(3):253-264.
[23]Strauer BE,Schannwell CM,Brehm M.Therapeutic potentials of stem cells in cardiac diseases.Minerva Cardioangiol,2009,57(2):249-267.
[24]Yen AH,Sharpe PT.Stem cells and tooth tissue engineering.Cell Tissue Res.2008,331(1):359-372.
[25]Centeno C J,Busse D,Kisiday J,Keohan C,Freeman M,Karli D.Regeneration of meniscus cartilage in a knee treated with percutaneously implanted autologous mesenchymal stem cells.Med.Hypotheses,2008,71(6):900-908.
[26]司徒镇强,吴军正.细胞培养[M].西安,世界图书出版社,1996,299-300.
[27]Daniel J M(u|¨)ller,Michael Krieg,David Alsteens and Yves F Dufre ^ ne.New frontiers in atomic force microscopy:analyzing interactions from single-molecules to cells,Current Opinion in Biotechnology,2009,20:4-13.
[1]Pierre Parotl,Yves F.Dufre(?)ne,Peter Hinterdorfer,et al.Past,present and future of atomic force microscopy in life sciences and medicine.J.Mol.Recognit,2007,20:418-431.
[2]X.Li,B.E.Logan,Analysis of Bacterial Adhesion Using a Gradient Force Analysis Method and Colloid Probe Atomic Force Microscopy.Langmuir 2004,20 (20):8817-8822.
[3]H.H.P.Fang,K.Y.Chan,L.C.Xu,Quantification of bacterial adhesion using atomic force microscopy.J Microbiol Methods,2000,40:89-98.
[4]K.Tsukada,E.Sekizuka,C.Oshio and H.Minamitani,Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system,Microvasc.Res.2001,61:231-239.
[5]L.O.Simpson,Intrinsic stiffening of red blood cells as the fundamental cause of diabetic nephropathy and microangiopathy,Nephron,1985,39:344-351.
[6]G M Artmann,K L Sung,T Horn,D Whittemore,G Norwich,and S Chien Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.Biophys J.1997 March;72(3):1434-1441.
[7]D.Koutsoris,R.Guillet,J.C.Lelievre,M.T.Guillemin,P.Bertholm,Y.Beuzard,M.Boynard,Determination of erythrocyte transit times through micropores.Ⅰ-Basic operational principles.Boynard,Biorheology 25 (1988)763-772.
[8]X.Wang,H.Zhao,F.Y.Zhuang,J.F.Stoltz.Measurement of erythrocyte deformability by two laser diffraction methods,Clinical Hemorheology and Microcirculation 21 (1999)291-295
[9]Brandao MM,Fontes A,Barjas-Castro ML,et al.Optical tweezers for measuring red blood cell elasticity:application to the study of drug response in sickle cell disease,Eur J Haematol 2003:70:207-211
[10]Andrew E.Pelling,Sadaf Sehati,Edith B.Gralla,et al.Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae,Science,2004,305:1147-1150.
[11]N.Babu and M.Singh,Influence of hyperglycemia on aggregation,deformability and shape parameters of erythroeytes,Clin.Hemorheol.Microcirc.2004,31:273-280.
[12]袁宝军,安新,王冬梅等,2型糖尿病患者红细胞CD35、CD44s和CD58分子含量的变化及临床意义,中国实验诊断学2005,9:744-746.
[13]Thirunavukkarasu V,Anitha Nandhini AT,Anuradha CV.Lipoic acid attenuates hypertension and improves insulin sensitivity,kallikrein activity and nitrite levels in high fructose-fed rats,J Comp Physiol,2004,174:587-592.
[14]张雪冰,白然,杜建玲等,Na/K ATP酶活性及其ATP1A1基因多态性与2型糖尿病周围神经病变的相关性,中国糖尿病杂志,2007,15:222-224.
[15]Miossec P,Zkhiri F,Paries J,et al.Effect of pravastatin on erythrocyte rheological and biochemical properties in poorly controlled type 2 diabetic patients,Diabet Med,1999,16:424-430.
[2]Muller DJ.AFM:a nanotool in membrane biology,Biochemistry,2008,47(31):7986-7998.
[3]Pierre Parot,Yves F.Dufre ^ne,Peter Hinterdorfer,Christian Le Grimellec,Daniel Navajas,Jean-Luc Pellequer and Simon Scheuring.Past,present and future of atomic force microscopy in life sciences and medicine.J.Mol.Recognit.2007,20:418-431.
[4]F.J.Rubio-Sierra,W.M.Heckl,R.W.Stark.Nanomanipulation by Atomic Force Microscopy,Advanced Engineering Materials,2005,7:193 - 196.
[5]C.Baur et al.,"Nanoparticle manipulation by mechanical pushing:underlying phenomena and real-time monitoring",Nanotechnology,1998,9(4):360-364.
[6]R.M.Taylor et al.,"The nanomanipulator:a virtual reality interface for a scanning tunneling microscope",Proc.ACM SIGGRAPH,Anaheim,CA,1993,8:127-134.
[7]G.Binning,C.F.Quate,Ch.Gerber.Atomic force microscope.Phys.Rev.Lett.1986,56(9):930.
[8]BALDWIN,P.M,FRAZIER,R.A,ADLER,J,GLASBEY,T.O,KEANE,M.P,ROBERTS,C.J,TENDLER,S.J.B,DAVIES,M.C,MELIA,C.D.Surface imaging of thermally sensitive particulate and fibrous materials with the atomic force microscope:a novel sample preparation method.Journal of Microscopy.1996,184(2):75-80.
[1]Ana P.Castano,Pawel Mroz,Michael R.Harnblin.Photodynarnic therapy and anti-tumour Immunity,Nat Rev Cancer.2006,6:535-545.
[2]唐蕾,诸葛健.天然光敏剂的光动力杀伤作用[J].中国抗生素杂志,2004,29(11):703-706.
[3]Yoshimura M.,Namura S.,Akamatsu H.and Horio T..Antimicrobial effects of phototherapy and photochemotherapy in vivo and in vitro[J].British Dermatology,1996,135:528-532.
[4]M.Wainwright,Photoantimicrobials - a PACT against resistance and infection,Drugs of the Future,2004,29:85-93.
[5]Cecilia Bombelli,Federico Bordi,Stefania Ferro,et al.New Cationic Liposomes as Vehicles of m-Tetrahydroxyphenylchlorin in Photodynamic Therapy of Infectious Diseases Mol.Pharmaceutics,2008,5(4),672-679
[6]Tim Maisch.Anti-microbial photodynamic therapy:,useful in the future? Lasers Med Sci,2007,22:83-91.
[7]FOOTE C S.Type Ⅰ and Type Ⅱ Mechanisms of Photodynamic Action.In Light Activeted Pesticides[M].Wastington:American Chemical Society,1987,22228.
[8]Nitzan Y.,Gutterman M.and Malik Z..Inactivation of gram negative bacteria by photosensitized porphyrins[J].Photochemistry and Photobiology.1992,55:89 -96.
[9]Saeki Y.,Ito Y.and Shibata M.l..Antimicrobial action of green tea extract,flavono flavor and copper chlorophyll against oral bacteria[J].Bull Tokyo DentColl,1993,34(1):33-37.
[10]North J.,Neyndorff H.and Levy J.G..Photosensitizers as virucidal agents[J].Journal of Photochemistry and Photobiology.B:Bioogyl,1993,17(2):99-108.
[11]Malik Z.,Ladan H.and Niton Y..The bactericidal activity of a deuteroporphyrin hemin mixture on grampostive bacteria.A microbiological and spectroscopic study[J].Journal of Photochemistry and photobiology,1990,B(6):419 - 430.
[12]陈文晖,余建鑫,姚建忠,沈卫镝,刘建飞,许德余.光动力治癌药血啉甲醚的药代动力学研究[J].中国激光医学杂志,2000,9(2):106-108.
[13]瞿燕,韩光范.光动力学疗法治癌光敏剂的研究进展[J].中国激光医学杂志,2006,15(1):48-55.
[14]戴维德,王雷,刘凡光,顾瑛,李晓松,丁新民,曾晶.应用激光扫描共聚焦显微成像术研究光敏剂亚细胞定位[J].中国激光医学杂志,2004,13(1):12-17.
[15]顾瑛,李峻亨,王开,江亿平,梁洁,朱建国.血啉甲醚用于光动力疗法治疗鲜红斑痣的初步临床研究[J].中国激光医学杂志,1996,5(4):201-204.
[16]郑蔚,谢树森,陆祖康.用激光荧光法诊断和定位早期肺癌[J].激光与光电子学进展,1998,(6):29-40.
[17]王伟,李辉,李家泽,尚立群,刘小兵.肺癌组织血卟啉单甲醚激光诱发药物荧光与激光诱发自体荧光的光谱区别[J].中国激光医学杂志2003,12(4):243-246.
[18]胡韶山,王齐,岳武,杨宝峰.光动力辅助显微手术治疗脑胶质瘤[J].中华神经外科杂志,2004,20(1):30-32.
[19]Xinmin Ding,,Qinzhi Xu,Fanguang Liu,Pingkun Zhou,Ying Gu,Jing Zeng,Jing An,Weide Dai,Xiaosong Li.Hematoporphyrin monomethyl ether photodynamic damage on HeLa cells by means of reactive oxygen species production and cytosolic free calcium concentration elevation,Cancer Letters,2004,216:43-54.
[20]武汉大学生物系微生物教学研究小组.微生物[M].北京科学出版社.1987.61-64.
[21]张文治.新编食品微生物学[M].中国轻工业出版社1995,5:10-23.
[22]牛天贵.张宝芹.食品微生物检验[M].中国计量出版社,2003,2:252-312.
[23]周德庆.微生物学实验教程[M].北京:高等教育出版社,2006,109-113
[24]Malik,Z.,Ladan,H.and Nitzan,Y..Photodynamic inactivation of Gram negative bacteria:problems and possible solutions[J].Photochemistry and Photobiology,1990,5(B):811-816.
[25]郑瑜谦,闫福华,陈锦灿,赵欣,李永东,袁彩,石晓莉,黄明东.一种新型光敏剂对牙周致病菌的体外抗菌研究.口腔医学研究,2006,22(4):368-371.
[26]Taylor PW,Stapleton PD,Luzio P.New ways to treat bacterial infections.Drug Discov Today,2002,7:1086-1091.
[27]Nitzan Y,Guttennan M,Malik Z,et al.Inactivation of gram-negative bacteria by photosensitized porphyrins.Photochem Photobiol,1992,55:89-96.
[28]Maisch T,Szeimies RM,Jori G,et al.Antibacterial photodynamic therapy in dermatology.Photochem Photobiol Sci,2004,3:907-917.
[29]Menezes S,Capella MA,Caldas LR.Photodynamic action of methylene blue:repair and mutation in Escherichia coli.J Photochem Photobiol B.1990,5:505-517.
[30]J.M.Bliss,C.E.Bigelow,T.H.Foster and C.G.Haidaris,Susceptibility of Candida species to photodynamic effects of Photofrin,Antimicrob Agent Chemother,2004,48:2000-2006.
[1]陈宜张,林其谁主编.生命科学中的单分子行为及细胞内实时检测[M].北京:科学出版社,2005,1-11.
[2]Pfister G,Stroh CM,Perschinka H,et al.Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.J Cell Sci,2005,118(8):1587-94.
[3]Puntheeranurak T,Wildling L,Gruber HJ,et al.Ligands on the string:single-molecule AFM studies on the interaction of antibodies and substrates with the Na+-glucose co-transporter SGLT1 in living cells.J Cell Sci,2006,119(14):2960-2967.
[4]Kim H,Arakawa H,Hatae N,et al.Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM.Ultramicroscopy,2006,106(8-9):652-662.
[5]Madl J,Rhode S,Stangl H,et al.A combined optical and atomic force microscope for live cell investigations.Ultramicroscopy,2006,106(8-9):645-651.
[6]Hong Wang,Ingrid Tessmer,Deborah L.Croteau,Dorothy A.Erie,and Bennett Van Houten.Functional Characterization and Atomic Force Microscopy of a DNA Repair Protein Conjugated to a Quantum Dot,Nano Lett,2008,8(6):1631-1637.
[7]Peter Scho "n,,Martin Go " rlich,Michiel J.J.Coenen,Hans A.Heus,and Sylvia Speller.Nonspecific Protein Adsorption at the Single Molecule Level Studied by Atomic Force Microscopy,Langmuir,2007,23(20):9921-9923.
[8]White,S.H.,and Wimley,W.C.Membrane protein folding and stability:Physical principles.Annu.ReV.Biophys.Biomol.Struct.1999,28:319-365.
[9]Sapra,K.T.,Park,P.S.,Palczewski,K.,and Muller,D.J.Mechanical properties of bovine rhodopsin and bacteriorhodopsin:Possible roles in folding and function.Langmuir,2008,24:1330-1337.
[10]Scheuring,S.,Seguin,J.,Marco,S.,Levy,D.,Robert,B.,and Rigaud,J.L.Nanodissection and high-resolution imaging of the Rhodopseudomonas Viridis photosynthetic core complex in native membranes by AFM.Proc.Natl.Acad.Sci. U.S.A.2003,100:1690-1693.
[11]Alattia,J.R.,Shaw,J.E.,Yip,C.M,and Prive,G G.Molecular imaging of membrane interfaces reveals mode of P-glucosidase activation by saposin C.Proc.Natl.Acad.Sci.U.S.A.2008,104:17394-17399.
[12]Monia Taranta,Anna Rita Bizzar and Salvatore Cannistraro.Probing the interaction between p53 and the bacterial protein azurin by single molecule force spectroscopy,J.Mol.Recognit.2008,21:63-70.
[13]Berquand A,Xia N,Castner DG,Clare BH,Abbott NL,Dupres V,Adriaensen Y,Dufrene YF.Antigen bindingforces of single antilyso-zyme Fv fragments explored by atomic force microscopy.Langmuir,2005,21:5517-5523.
[14]Schwesinger F,Ros R,Strunz T,Anselmetti D,Guntherodt H-J,Honegger A,Jermutus L,Tiefenauer L,Pluckthun A.Unbinding forces of single antibody-antigen complexes correlatewith their thermal dissociation rates.Proc.Natl.Acad.Sci.U S A,2000,97:9972-9977.
[15]Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell,2006,126 (4):663-76.
[16]Barrilleaux B,Phinney DG,Prockop DJ,O'Connor KC."Review:ex vivo engineering of living tissues with adult stem cells".Tissue Eng,2006,12 (11):3007-3019.
[17]Beckmann J,Scheitza S,Wernet P,Fischer JC,Giebel B.“Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment:identification of asymmetrically segregating proteins”.Blood,2007,109 (12):5494-5501.
[18]Adewumi O,Aflatoonian B,Ahrlund-Richter L,et al.“Characterization of human embryonic stem cell lines by the International Stem Cell Initiative”.Nat.Biotechnol,2007,25 (7):803-816.
[19]Kazuhiro Sudo,Megumi Kanno,Kenichi Miharada,Saeri Ogawa,Takashi Hiroyama,Kaoru Saijo,Yukio Nakamura.Mesenchymal Progenitors Able to Differentiate into Osteogenic,Chondrogenic,and/or Adipogenic Cells In Vitro Are Present in Most Primary Fibroblast-Like Cell Populations.Stem Cells,2007,25(7):1610 -1617.
[20]Bruder SP,Fink D J,Caplan AI.Mesenchymal stem cells in bone development,bone repair,and skeletal regeneration therapy.J Cell Biochem.1994,56(3):283-294.
[21]Bielby R,Jones E,McGonagle D.The role of mesenchymal stem cells in maintenance and repair of bone.Injury,2007,38:S26-32.
[22]Chotima Boettcher.Gene-modified bone marrow-derived stem cells:an attractive gene delivery system in inherited retinal disorders,Asian Biomedicine 2007,1(3):253-264.
[23]Strauer BE,Schannwell CM,Brehm M.Therapeutic potentials of stem cells in cardiac diseases.Minerva Cardioangiol,2009,57(2):249-267.
[24]Yen AH,Sharpe PT.Stem cells and tooth tissue engineering.Cell Tissue Res.2008,331(1):359-372.
[25]Centeno C J,Busse D,Kisiday J,Keohan C,Freeman M,Karli D.Regeneration of meniscus cartilage in a knee treated with percutaneously implanted autologous mesenchymal stem cells.Med.Hypotheses,2008,71(6):900-908.
[26]司徒镇强,吴军正.细胞培养[M].西安,世界图书出版社,1996,299-300.
[27]Daniel J M(u|¨)ller,Michael Krieg,David Alsteens and Yves F Dufre ^ ne.New frontiers in atomic force microscopy:analyzing interactions from single-molecules to cells,Current Opinion in Biotechnology,2009,20:4-13.
[1]Pierre Parotl,Yves F.Dufre(?)ne,Peter Hinterdorfer,et al.Past,present and future of atomic force microscopy in life sciences and medicine.J.Mol.Recognit,2007,20:418-431.
[2]X.Li,B.E.Logan,Analysis of Bacterial Adhesion Using a Gradient Force Analysis Method and Colloid Probe Atomic Force Microscopy.Langmuir 2004,20 (20):8817-8822.
[3]H.H.P.Fang,K.Y.Chan,L.C.Xu,Quantification of bacterial adhesion using atomic force microscopy.J Microbiol Methods,2000,40:89-98.
[4]K.Tsukada,E.Sekizuka,C.Oshio and H.Minamitani,Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system,Microvasc.Res.2001,61:231-239.
[5]L.O.Simpson,Intrinsic stiffening of red blood cells as the fundamental cause of diabetic nephropathy and microangiopathy,Nephron,1985,39:344-351.
[6]G M Artmann,K L Sung,T Horn,D Whittemore,G Norwich,and S Chien Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.Biophys J.1997 March;72(3):1434-1441.
[7]D.Koutsoris,R.Guillet,J.C.Lelievre,M.T.Guillemin,P.Bertholm,Y.Beuzard,M.Boynard,Determination of erythrocyte transit times through micropores.Ⅰ-Basic operational principles.Boynard,Biorheology 25 (1988)763-772.
[8]X.Wang,H.Zhao,F.Y.Zhuang,J.F.Stoltz.Measurement of erythrocyte deformability by two laser diffraction methods,Clinical Hemorheology and Microcirculation 21 (1999)291-295
[9]Brandao MM,Fontes A,Barjas-Castro ML,et al.Optical tweezers for measuring red blood cell elasticity:application to the study of drug response in sickle cell disease,Eur J Haematol 2003:70:207-211
[10]Andrew E.Pelling,Sadaf Sehati,Edith B.Gralla,et al.Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae,Science,2004,305:1147-1150.
[11]N.Babu and M.Singh,Influence of hyperglycemia on aggregation,deformability and shape parameters of erythroeytes,Clin.Hemorheol.Microcirc.2004,31:273-280.
[12]袁宝军,安新,王冬梅等,2型糖尿病患者红细胞CD35、CD44s和CD58分子含量的变化及临床意义,中国实验诊断学2005,9:744-746.
[13]Thirunavukkarasu V,Anitha Nandhini AT,Anuradha CV.Lipoic acid attenuates hypertension and improves insulin sensitivity,kallikrein activity and nitrite levels in high fructose-fed rats,J Comp Physiol,2004,174:587-592.
[14]张雪冰,白然,杜建玲等,Na/K ATP酶活性及其ATP1A1基因多态性与2型糖尿病周围神经病变的相关性,中国糖尿病杂志,2007,15:222-224.
[15]Miossec P,Zkhiri F,Paries J,et al.Effect of pravastatin on erythrocyte rheological and biochemical properties in poorly controlled type 2 diabetic patients,Diabet Med,1999,16:424-430.