上皮损伤制作分析<em>阿米巴</em>感染
1Department of Infectomics and Molecular Pathogenesis,Center for Research and Advanced Studies of the National Polytechnic Institute, 2Department of Molecular Biomedicine,Center for Research and Advanced Studies of the National Polytechnic Institute, 3Agency for Knowledge Commercialization,Center for Research and Advanced Studies of the National Polytechnic Institute
Summary
人感染阿米巴导致阿米巴病，腹泻在热带国家的重要原因。感染是通过与肠上皮细胞病原体相互作用启动，引起的细胞 – 细胞接触的开口，因而腹泻，有时接着肝感染。本文提供了一个模型来评估早期的宿主 – 病原体相互作用，以改善我们的阿米巴病发病机制的认识。
Abstract
阿米巴是人类阿米巴病的病原体，腹泻和肝脓肿在热带国家的重要原因。感染是通过与肠上皮细胞的病原体的相互作用启动。这种相互作用导致细胞间的结构，例如紧密连接（TJ）的中断。 TJ保证上皮细胞层，以宿主组织从肠腔中分离的密封。最近的研究提供的证据表明由寄生蛋白EhCPADH112中断TJ的是大肠杆菌的先决条件阿米巴入侵是伴随着上皮屏障功能障碍。因此， 大肠杆菌中参与TJ拆卸分子机制的分析阿米巴入侵是非常重要的改善我们的阿米巴病发病机制的认识。本文介绍了一个简单的模型，它允许初始宿主 – 病原体相互作用的评估和寄生虫侵袭潜能。要分析的参数包括Transepithelial电阻，EhCPADH112与上皮细胞表面的受体，改变表达与上皮交界标记和上皮细胞内的寄生虫分子的本地化本地化互动。
Introduction
阿米巴是一种单细胞原生动物负责人阿米巴病，肠道感染引起炎症和腹泻。E.阿米巴感染高达50亿人每年，但只有约10％的感染者制定相关的阿米巴病1，症状。感染发生于对含有大肠杆菌污染的食物或水摄入阿米巴包囊。在肠道内，囊肿产生坚持结肠粘蛋白和增殖2现场滋养体。滋养体通常形成是通过粪便排出体外囊肿。在其他情况下和未知的原因，滋养体打破肠上皮细胞层和侵入皮下组织。在最坏的情况下，他们进入血流而影响其他器官，如肝3。
打破上皮屏障需要保持细胞加入transmembranal上皮结构的破坏。上皮细胞触点由根尖连接复合体组成的紧（TJ）形成和粘合连接（AJ）和桥粒4。最顶端的交界处TJ，因此，它们是由E.冒犯的第一阻挡主机入侵期间组织阿米巴和一些其他病原体。 TJ是由transmembranal粘附受体如claudins，occludin和连接粘附分子（JAM）从事均聚物或嗜异性的相互作用与所述相邻小区的受体。它们是由紧密连接（ZO）系列连接粘附受体与肌动蛋白细胞骨架，以提供进一步的机械强度上皮的支架分子在细胞内的约束。 TJ负责密封肠组织从肠腔，防止过多的水分和溶质渗漏。因此，后TJ由寄生虫破坏，组织被入侵。E.阿米巴分泌一些分子，如：（ⅰ）涉及变形虫的粘附性尔格等细胞5; （ii）在胞吐作用参与杀伤宿主细胞的膜活性的因素，例如被称为离子通道形成肽amoebapores 6,7;及（iii）降解细胞外基质蛋白并介导组织崩解5,8,9蛋白酶。
半胱氨酸蛋白酶EhCP112和粘附分子EhADH112，它们一起组成EhCPADH112复杂两种E.阿米巴毒性蛋白，在TJ 10的拆装发挥了重要作用。现场滋养体，他们的总裂解液和分泌产物诱导的上皮屏障的TJ复杂和功能障碍的分子变化。在这项研究中，它表明，EhCP112和EhADH112与occludin和紧密连接蛋白-1的蛋白质，导致内在化和细胞蛋白的降解相互作用，从而有利于E。通过旁细胞途径阿米巴入口。
我们的数据和那些ØF其他群体11-17强烈建议特定的宿主-病原体相互作用，使寄生虫侵袭的必要性。揭开这些相互作用的分子基础是最重要的一个更好的了解阿米巴病的发病机制。 TJ由滋养体，其特征在于，增加细胞旁渗透性选择性的干扰，可以通过在跨上皮电阻下降（TER）来测量。寄生蛋白对宿主上皮细胞的转移可以通过免疫荧光染色和共聚焦激光显微镜，也可以揭示共定位阿米巴毒力因子的上皮交界性标志物，指示可能的直接的相互作用的方法来确定。在这篇文章中，我们将详细介绍上皮细胞和滋养体如何栽培，收获和操纵研究宿主 – 病原体相互作用及其后果。
Protocol
1，E.建立和维护阿米巴文化成长axenically（完全免费所有其他污染微生物）1×10 5滋养体阿米巴株HML的：IMSS克隆18在16 x 125毫米培养管聚四氟乙烯内衬螺旋盖（或1×10 6滋养体在一次性的T- 25烧瓶中）和15毫升（或50毫升缇-S-33培养基中的T-25烧瓶中）（缇肉汤补充有3％的钻石维生素混合物，10％热灭活的成年牛血清，0.5国际单位/毫升青霉素和35微克/?…
Representative Results
对于一个成功的E。阿米巴文化，有两个重要条件必须满足：成长在无菌条件下与收获对数生长期。 E.以前，文化阿米巴是容易建立与细菌或锥虫22的某些物种的关联。但是，现在它通常有无菌培养这种寄生虫的意思变形虫在免费代谢细菌，真菌，原生动物，后生动物或细胞环境中的不确定传代培养。此外，后期对数生长早期稳定期收获滋养体关键是有活力和增殖细胞<s…
Discussion
为了研究上皮感染时体外宿主-病原体相互作用的E.组织阿米巴 ，关键的是与两个上皮细胞和滋养体行之有效的培养工作。例如，以前，E.阿米巴培养了通常建立在与细菌或锥虫22,23某些物种的关联。然而，共培养的大肠杆菌阿米巴培养是得不偿失的宿主-病原体相互作用的研究，因为在宿主细胞中观察到的效果不能明确归因于ameobas但可以相当是共培养细胞的效果?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the Institute of Science and Technology of the Federal District (ICyTDF, 64/2012 to EO) and the Mexican Council for Science and Technology (Conacyt, 179895 to MS).
Materials
| Entamoeba histolytica HM1:IMSS, Clone A | IMSS Hospital, Mexico | Without/number | Virulent trophozoites18 |
| TYI broth | Becton, Dickinson and Company Merck Merck Merck J.T. Baker Reproquifin SIGMA-Aldrich SIGMA-Aldrich |
211862 K35625437 626 21578 4873 3252-01 CAS 50-81-7 C7880 F-5879 |
3.45% BBL Biosate peptone 58 mM glucose 39 mM NaCl 5 mM KH2PO4 6.5 mM K2HPO4 16.3 mM ascorbic acid 8.1 mM L-cysteine 0.1 mM ferric ammonium citrate, adjust pH 6.819 |
| Bovine serum adult | Microlab , Labs., Mex. | SU146 | Use at 10% and inactivated to 56° C for 30 min |
| Diamond vitamin mixture- Tween 80 | In vitro | SR-07 | Use at 3% |
| Penicillin | Lakeside, Méx. | 34564SSA IV | 0.5 IU/mL |
| Streptomycin | Lakeside, Méx. | 75757SSA IV | 35 µg/ mL |
| Pyrex 15 mL screw cap culture tubes with PTFE lined phenolic caps | Corning-Pyrex | 9826-16X | 16×125 mm, capacity 15 mL and caps fabricated from special formula resistant to effects of temperature |
| Cell culture plates, 6 Well | Corning-Costar | 3516 | Sterile plates, well diameter 34.8 mm and growth area 9.5 cm2. Rings on lid prevent cross-contamination |
| 25cm2 cell culture flask | Corning-Costar | 430168 | Canted neck flasks |
| MDCK (Madin Darby canine kidney) type I | American Type Culture Collection | CCL34 | Kidney epithelial cells grown between the 60th and 90th passage |
| DMEM medium | Gibco | 12800-017 | Dulbecco's Modified Eagle Medium with high glucose. |
| Neonate Calf Serum | In vitro | S-02 | Use at 10%. |
| Penicillin/Streptomycin mixture | In vitro | A-01 | Stock solution 10,000 U/µg/mL |
| Insulin | AMSA | 398MJ94SSA IV | Stock solution 100 IU/mL |
| Trypsin solution | In vitro | EN-005 | 0.05% enzyme solution without calcium and magnesium |
| 75cm2 cell culture flask | Corning-Costar | 430720 | Canted neck flasks for trophozoite culture in TYI-S-33 medium |
| Transwell permeable supports | Corning-Costar | 3470 | 0.4. µm polyster membrane, 6.5 mm insert in 24 well plate, growth area 0.3 cm2 |
| 24 well cell culture dish | Corning-Costar | 3524 | Clear polystyrene, treated for optimal cell attachment, sterilized by gamma radiation and certified non-pyrogenic |
| Complete Mini | Roche | 11836 153 001 | Protease inhibitor cocktail inhibits a broad spectrum of serine, cysteine and metallo-proteases. Final concentration 1 mM |
| Trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64) | SIGMA-Aldrich | E3132 | Cystein protease inhibitor, final concentration 40 µg/mL |
| pαZO-1 | Invitrogen | 402200 | IgG rabbit policlonal antibody against a synthetic peptide in the middle region of the ZO-1 human protein |
| mαEhCPADH112 | Homemade antibody | Without/ Number | IgM mouse monoclonal antibody against 444-601 epitope located at C-terminal of EhCPADH11221,27 |
| FITC-goat anti-mouse IgM | Zymed | 62-6811 | Fluorescein isotiocyanate (FITC)-labelled goat anti-mouse secondary antibody |
| TRITC- goat anti-rabbit IgG (H+L) | Zymed | 816114 | Tetramethyl-rhodamine isothiocyanate (TRITC)-labelled goat anti-rabbit IgG secondary antibody. |
| STX2 Electrode | World Precision Instrument | 102711 | Consists of a fixed pair of double electrodes, 4 mm wide and 1 mm thick. Each stick of the electrode pair contains a silver/silver-chloride pellet for measuring voltage and a silver electrode for passing current. For use with EVOM |
| EVOM epithelial voltohmmeter | World Precision Instrument | 12111 | Use in resistance mode and maintain unplugged during TER measurements |
| Neubauer chamber | MEARIENFELD | 610610 | Hemocytometer |
| Leica TCS_SP5_MO | Leica | Without/number | Laser confocal microscopy with Leica microsystems CMS Gmbh/leica Las af Lite/BIN software |
| Vectashield | Vector Laboratories, Inc. | H-1000 | Mounting medium for fluorescence |
| 4´, 6-diamino-2-phenylindole (Dapi) | SIGMA | D-9542 | 0.05 mM final concentration |
| Bovine serum albumin (BSA) | US Biological | A-1310 | 0.5% final concentration for blocking solution |
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Citer Cet Article
Betanzos, A., Schnoor, M., Javier-Reyna, R., García-Rivera, G., Bañuelos, C., Pais-Morales, J., Orozco, E. Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection. J. Vis. Exp. (88), e51668, doi:10.3791/51668 (2014).