幽门螺杆菌感染与胃病理的小鼠模型
Summary
小鼠是研究胃肠道微生物感染和疾病的一种宝贵的体内模型。本文介绍了研究幽门螺杆菌相关疾病小鼠模型细菌定植和组织病理学变化的方法。
Abstract
幽门螺杆菌是存在于全球半数人口中的一种胃病原体, 是人类发病率和死亡率的一个重要原因。开发了几种小鼠胃幽门螺杆菌感染模型, 研究了幽门螺旋菌在人体宿主胃中的分子和细胞机制, 并引起疾病。在这里, 我们描述的协议: 1) 通过胃内灌胃制备小鼠体内感染的细菌悬浮液;2) 通过聚合酶链反应 (PCR) 和可行计数确定小鼠胃组织细菌定植水平;3) 通过组织学评估病理变化。为了建立小鼠Helicobacter 感染, 特定的无病原体 (SPF) 动物首先接种悬浮液 (包含≥105菌落形成单位, CFUs) 的小鼠殖民菌株的幽门螺杆菌或幽门螺杆菌等动物的其他胃幽门螺杆菌河猫。在适当的时间点后感染, 胃被切除和解剖弧矢成两个相等的组织片段, 每个组成的窦和身体区域。其中一个片段, 然后用于可行计数或 DNA 提取, 而另一种是受到组织学处理。在胃组织切片染色与沃辛瘤-星光, 姬姆萨或 Haematoxylin 和伊红 (H & E) 污渍的情况下, 可能定期评估胃肠细菌的殖民化和组织学变化。其他免疫学分析也可以通过免疫组化或免疫荧光法对小鼠胃组织切片进行。下面所述的协议是专门设计的, 能够对类似于人体相关的幽门螺杆菌疾病的小鼠进行评估, 包括炎症、腺体萎缩和淋巴毛囊形成。接种剂的制备和胃内灌胃协议也可用于研究其他肠道人类病原体, 如鼠伤寒沙门氏菌或柠檬酸 rodentium的殖民化老鼠的发病机制。
Introduction
幽门螺杆菌是一种螺旋状, 革兰阴性, 人胃病原体存在于世界各地的所有人群中, 发展中国家的感染率估计为 80%1。虽然大多数幽门螺杆菌感染的个体是无症状的, 一些发展更严重的疾病, 从消化性溃疡到胃癌2。幽门螺杆菌相关的癌症广泛的特点要么是恶性变化的上皮细胞 (风险) 或形成的额外淋巴结淋巴组织在胃, 导致胃腺癌或粘膜相关的淋巴组织 (麦芽) 淋巴瘤, 分别。幽门螺杆菌是高度适应的生存在恶劣的生态利基的胃由于存在各种致病因素和机制, 促进其在这个利基的坚持, 生长和新陈代谢。特别是,幽门螺杆菌的毒株具有 40 kb cag致病性岛 (cag), 用于编码生产4型分泌系统所需的大约30个基因 (T4SS)3,4.cag排阳性幽门螺杆菌菌株与宿主中更高水平的慢性炎症有关, 这已被牵连为胃腺癌5的重要前兆。
在体内动物模型, 特别是小鼠, 通过允许研究人员调查宿主、细菌和环境因素对幽门螺杆菌感染和疾病结果6的相对贡献, 获得了高度的信息。研究表明, 长时间幽门螺杆菌感染的小鼠在 C57BL/6 的遗传背景下, 导致慢性胃炎和腺萎缩的发展, 这两个特点是幽门螺杆菌感染7。此外, 感染与相关的猫/犬细菌种类,河猫, 已被证明诱导麦芽形成在类似的病理和疾病进展的小鼠在人类麦芽淋巴瘤8,9。在小鼠殖民学研究中最常用的幽门螺杆菌是 “悉尼菌株 1” (SS1) 菌株10, 这是cagPAI+但有一个非功能性 T4SS (T4SS−)11。其他广泛使用的菌株包括幽门螺杆菌B128 7.13 (cag排+/T4SS+)12和 X47-2AL (cag–/T4SS−)13。对于H. 河猫感染, 应变 CS1 (“Cat 螺旋 1”, cagT4SS−) 通常使用14.
在这里, 我们提供了一个协议, 描述了在体内感染的幽门螺菌剂的制备, 小鼠胃内灌胃的程序, 以及组织的处理方法病理学改变的研究在胃里。特别是, 本文将重点介绍用于形象化细菌殖民化的组织学方法, 并评估感染小鼠胃黏膜中的组织学变化, 包括麦芽的形成。这里描述的一些方法可以适应其他肠道病原体的研究, 如S.鼠伤寒或rodentium。
Protocol
1. 细菌菌剂的生长与制备 将幽门螺杆菌或h. 河猫15的甘油从-80 摄氏度解冻, 并在马血琼脂 (HBA) 板上进行亚型, 包括: 血琼脂碱 2 (见材料表);一种改良的 “Skirrow 抗生素选择性补充剂” (包括万古霉素, 10 微克/毫升; 多粘菌 b, 25 ng/毫升; 甲氧苄啶, 5 µg/毫升; 两性不变性 b, 2.5 微克/毫升);和 5–10% (v/v) 马血15,16?…
Representative Results
本协议描述的口服灌胃技术, 以实现胃内感染与幽门螺杆菌或河猫在小鼠模型 (图 1)。安乐死后, 胃被移除, 称重并分为2个相等的一半, 包括胃组织的窦、体和非腺区域 (图 2)。在进行任何分析之前删除非腺区域。 通过对幽门螺杆菌感染的胃匀浆进行可行计数, 并随后在 HBA 板上枚举单个菌落 (图 3</…
Discussion
本协议描述了对幽门螺杆菌感染的体内小鼠模型的使用。该程序的关键步骤是: 1) 制备含有活性和能动细菌的幽门菌剂;2) 通过胃内灌胃向鼠标提供适当数量的细菌;3) 通过菌落计数和/或 PCR 检测细菌感染;和 4) 胃组织的处理, 以便评估受感染的胃的病理组织学。下面将讨论有关修改、故障排除和技术考虑的进一步建议。
在我们的实验室中, 使用血琼脂基2号补…
Disclosures
The authors have nothing to disclose.
Acknowledgements
提交人谨感谢保利女士和乔治雷-麦肯女士提供技术援助。作者承认, 莫纳什大学解剖学和发育生物学系的莫纳什组织学平台的设施和技术援助的使用。该实验室由国家卫生和医学研究理事会 (澳洲) 向 RLF (APP1079930、APP1107930) 提供资金支持。RLF 由澳洲 (APP1079904) 的高级研究奖学金支持。KD 和 MC 都得到了莫纳什研究生奖学金的支持。KD 还得到了美国哈得逊医学研究所的先天免疫和传染性疾病中心的支持, 而 MC 拥有莫纳什大学医学、护理和健康科学学院的国际研究生奖学金。在哈得逊医学研究所的研究得到了维多利亚政府的运营基础设施支持计划的支持。
Materials
| Bacteriological reagents | |||
| Oxoid Blood Agar Base No.2 | Thermo Fischer Scientific | CM0271B | Dissolve in deinonized water prior to sterilization |
| Premium Defibrinated Horse blood | Australian Ethical Biologicals | PDHB100 | |
| Bacto Brain Heart Infusion Broth | BD Bioscience | 237500 | Dissolve in deinonized water prior to sterilization |
| CampyGen gas packs | Thermo Fischer Scientific | CN0035A/CN0025A | |
| Histological reagents | |||
| Formalin, neutral buffered, 10% | Sigma Aldrich | HT501128 | |
| Absolute alcohol, 100% Denatured | ChemSupply | AL048-20L-P | |
| Isopropanol (2-propanol) | Merck | 100995 | |
| Xylene (sulphur free) | ChemSupply | XT003-20L | |
| Mayer's Haematoxylin | Amber Scientific | MH-1L | Filter before use |
| Eosin, Aqueous Stain | Amber Scientific | EOCA-1L | Filter before use |
| Wright-Giemsa Stain, modified | Sigma Aldrich | WG80-2.5L | Dilute before use (20% Giemsa, 80% deionized water) |
| Histolene | Grale Scientific | 11031/5 | |
| DPX mounting medium | VWR | 1.00579.0500 | |
| Molecular biology reagents | |||
| Qubit dsDNA BR Assay Kit | Thermo Fischer Scientific | Q32850 | |
| Oligonucleotides | Sigma Aldrich | The annealing temperature of ureB primers used in this study is 61°C | |
| GoTaq Flexi DNA Polymerase | Promega | M8291 | Kit includes 10X PCR buffer and Magnesium Chloride |
| dNTPs | Bioline | BIO-39028 | Dilute to 10mM in sterile nuclease free water before use |
| Molecular Grade Agarose | Bioline | BIO-41025 | |
| Sodium Hydrogen Carbonate | Univar (Ajax Fine Chemicals) | A475-500G | |
| Magnesium Sulphate Heptahydrate | Chem-Supply | MA048-500G | |
| Antibiotics | |||
| Vancomycin | Sigma Aldrich | V2002-1G | Dissolve in deionized water |
| Polymyxin B | Sigma Aldrich | P4932-5MU | Dissolve in deionized water |
| Trimethoprim (≥98% HPLC) | Sigma Aldrich | T7883 | Dissolve in 100% (absolute) Ethanol |
| Amphotericin | Amresco (Astral Scientific) | E437-100MG | Dissolve in deionized water |
| Bacitracin from Bacillus licheniformis | Sigma Aldrich | B0125 | Dissolve in deionized water |
| Naladixic acid | Sigma Aldrich | N8878 | Dissolve in deionized water |
| Other reagents | |||
| Methoxyflurane (Pentrhox) | Medical Developments International | Not applicable | |
| Paraffin Wax | Paraplast Plus, Leica Biosystems | 39601006 | |
| Equipment and plasticware | |||
| Oxoid Anaerobic Jars | Thermo Fischer Scientific | HP0011/HP0031 | |
| COPAN Pasteur Pipettes | Interpath Services | 200CS01 | |
| Eppendorf 5810R centrifuge | Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4°C | ||
| 23g precision glide needle | BD Bioscience | 301805 | |
| Parafilm M | Bemis, VWR | PM996 | |
| Portex fine bore polythene tubing | Smiths Medical | 800/100/200 | |
| Plastic feeding catheters | Instech Laboratories | FTP20-30 | |
| 1 ml tuberculin luer slip disposable syringes | BD Bioscience | 302100 | |
| Eppendorf micropestle for 1.2 – 2 mL tubes | Sigma Aldrich | Z317314 | Autoclavable polypropylene pestles used for stomach homogenization |
| GentleMACs Dissociator | Miltenyi Biotec | 130-093-235 | Use a pre-set gentleMACS Programs for mouse stomach tissue |
| M Tubes (orange cap) | Miltenyi Biotec | 30-093-236 | |
| Qubit Fluorometer | Thermo Fischer Scientific | Q33216 | |
| Sterile plastic loop | LabServ | LBSLP7202 | |
| Cold Plate, Leica EG1160 Embedding System | Leica Biosystems | Not applicable | |
| Tissue-Tek Base Mould System, Base Mold 38 x 25 x 6 | Sakura, Alphen aan den Rijn | 4124 | |
| Tissue-Tek III Uni-Casette System | Sakura, Alphen aan den Rijn | 4170 | |
| Microtome, Leica RM2235 | Leica Biosystems | ||
| Charged SuperFrost Plus glass slides | Menzel Glaser, Thermo Fischer Scientific | 4951PLUS4 |
References
- Goh, K. L., Chan, W. K., Shiota, S., Yamaoka, Y. Epidemiology of Helicobacter pylori infection and public health implications. Helicobacter. 16, 1-9 (2011).
- Montecucco, C., Rappuoli, R. Living dangerously: how Helicobacter pylori survives in the human stomach. Nature Reviews Molecular Cell Biology. 2 (6), 457-466 (2001).
- Akopyants, N. S., et al. Analyses of the cag pathogenicity island of Helicobacter pylori. Molecular Microbiology. 28 (1), 37-53 (1998).
- Censini, S., et al. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proceedings of the National Academy of Sciences of the United States of America. 93 (25), 14648-14653 (1996).
- Peek, R. M., Fiske, C., Wilson, K. T. Role of innate immunity in Helicobacter pylori-induced gastric malignancy. Physiological Reviews. 90 (3), 831-858 (2010).
- O’Rourke, J. L., Lee, A. Animal models of Helicobacter pylori infection and disease. Microbes and Infection. 5 (8), 741-748 (2003).
- Sakagami, T., et al. Atrophic gastric changes in both Helicobacter felis and Helicobacter pylori infected mice are host dependent and separate from antral gastritis. Gut. 39 (5), 639-648 (1996).
- Correa, P. Helicobacter pylori and gastric carcinogenesis. The American Journal of Surgical Pathology. 19, S37-S43 (1995).
- Enno, A., et al. MALToma-like lesions in the murine gastric mucosa after long-term infection with Helicobacter felis. A mouse model of Helicobacter pylori-induced gastric lymphoma. The American Journal of Pathology. 147 (1), 217-222 (1995).
- Lee, A., et al. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology. 112 (4), 1386-1397 (1997).
- Crabtree, J. E., Ferrero, R. L., Kusters, J. G. The mouse colonizing Helicobacter pylori strain SS1 may lack a functional cag pathogenicity island. Helicobacter. 7 (2), 139-140 (2002).
- Israel, D. A., et al. Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses. Journal of Clinical Investigation. 107 (5), 611-620 (2001).
- Fox, J. G., et al. Helicobacter pylori-induced gastritis in the domestic cat. Infection and Immunity. 63 (7), 2674-2681 (1995).
- Lee, A., Hazell, S. L., O’Rourke, J., Kouprach, S. Isolation of a spiral-shaped bacterium from the cat stomach. Infection and Immunity. 56 (11), 2843-2850 (1988).
- Ferrero, R. L., Wilson, J. E., Sutton, P. Mouse models of Helicobacter-induced gastric cancer: use of cocarcinogens. Methods in Molecular Biology. 921, 157-173 (2012).
- Ferrero, R. L., Thiberge, J. M., Huerre, M., Labigne, A. Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection. Infection and Immunity. 66 (4), 1349-1355 (1998).
- Blanchard, T. G., Nedrud, J. G. Laboratory maintenance of Helicobacter species. Current Protocols in Microbiology. , (2012).
- Kim, J. S., Chang, J. H., Chung, S. I., Yum, J. S. Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy. FEMS Immunology and Medical Microbiology. 31 (1), 41-46 (2001).
- Nedrud, J. G., et al. Lack of genetic influence on the innate inflammatory response to helicobacter infection of the gastric mucosa. Frontiers in Immunology. 3, 181 (2012).
- Cai, X., et al. Helicobacter felis eradication restores normal architecture and inhibits gastric cancer progression in C57BL/6 mice. Gastroenterology. 128 (7), 1937-1952 (2005).
- Ferrero, R. L., Labigne, A. Cloning, expression and sequencing of Helicobacter felis urease genes. Molecular Microbiology. 9 (2), 323-333 (1993).
- Stevenson, T. H., Castillo, A., Lucia, L. M., Acuff, G. R. Growth of Helicobacter pylori in various liquid and plating media. Letters in Applied Microbiology. 30 (3), 192-196 (2000).
- Uotani, T., Graham, D. Y. Diagnosis of Helicobacter pylori using the rapid urease test. Annals of Translational Medicine. 3 (1), 9 (2015).
- Riley, L. K., Franklin, C. L., Hook, R. R., Besch-Williford, C. Identification of murine helicobacters by PCR and restriction enzyme analyses. Journal of Clinical Microbiology. 34 (4), 942-946 (1996).
- Chaouche-Drider, N., et al. A commensal Helicobacter sp. of the rodent intestinal flora activates TLR2 and NOD1 responses in epithelial cells. PLoS One. 4 (4), e5396 (2009).
- Fox, J. G. Helicobacter bilis: bacterial provocateur orchestrates host immune responses to commensal flora in a model of inflammatory bowel disease. Gut. 56 (7), 898-900 (2007).
- McGee, D. J., et al. Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37. Antimicrobial Agents and Chemotherapy. 55 (6), 2897-2904 (2011).
- Viala, J., et al. Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nature Immunology. 5 (11), 1166-1174 (2004).
- Kong, L., et al. A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model. Clinical and Vaccine Immunology. 3 (1), 73-78 (1996).
- Ng, G., Every, A., McGuckin, M., Sutton, P. Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis. Gut Microbes. 2 (6), 358-360 (2011).
- Ferrero, R. L., Ave, P., Radcliff, F. J., Labigne, A., Huerre, M. R. Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions. The Journal of Pathology. 191 (3), 333-340 (2000).
Tags
Keywords:
Mouse ModelHelicobacter InfectionGastric PathologiesMicrobiotaInflammationCancerPathogenesisGastrointestinal PathogensBacterial InoculumH. PyloriH. FelisMouse ColonizationIn Vitro SubcultureBacterial ViabilityBacterial MotilityGastric InoculationMouse RestraintEsophageal CatheterizationBacterial Dose
Cite This Article
D’Costa, K., Chonwerawong, M., Tran, L. S., Ferrero, R. L. Mouse Models Of Helicobacter Infection And Gastric Pathologies. J. Vis. Exp. (140), e56985, doi:10.3791/56985 (2018).