Name: mouse models of helicobacter infection and gastric pathologies
Uploaded: 2018-10-18T13:00:00
Description: Watch this Scientific Journal Video about Mouse Models Of Helicobacter Infection And Gastric Pathologies at JoVE.com

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。

1. 细菌菌剂的生长与制备
<ol><li>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?...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+the+cag+pathogenicity+island+of+Helicobacter+pylori.">Analyses of the cag pathogenicity island of Helicobacter pylori.</a> Molecular Microbiology. 28 (1), 37-53 (1998).</li><li>Censini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cag,+a+pathogenicity+island+of+Helicobacter+pylori,+encodes+type+I-specific+and+disease-associated+virulence+factors.">cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.</a> Proceedings of the National Academy of Sciences of the United States of America. 93 (25), 14648-14653 (1996).</li><li>Peek, R. M., Fiske, C., Wilson, K. 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A mouse model of Helicobacter pylori-induced gastric lymphoma.</a> The American Journal of Pathology. 147 (1), 217-222 (1995).</li><li>Lee, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+mouse+model+of+Helicobacter+pylori+infection:+introducing+the+Sydney+strain.">A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.</a> Gastroenterology. 112 (4), 1386-1397 (1997).</li><li>Crabtree, J. E., Ferrero, R. L., Kusters, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+colonizing+Helicobacter+pylori+strain+SS1+may+lack+a+functional+cag+pathogenicity+island.">The mouse colonizing Helicobacter pylori strain SS1 may lack a functional cag pathogenicity island.</a> Helicobacter. 7 (2), 139-140 (2002).</li><li>Israel, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori+strain-specific+differences+in+genetic+content,+identified+by+microarray,+influence+host+inflammatory+responses.">Helicobacter pylori strain-specific differences in genetic content, identified by microarray, influence host inflammatory responses.</a> Journal of Clinical Investigation. 107 (5), 611-620 (2001).</li><li>Fox, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+pylori-induced+gastritis+in+the+domestic+cat.">Helicobacter pylori-induced gastritis in the domestic cat.</a> Infection and Immunity. 63 (7), 2674-2681 (1995).</li><li>Lee, A., Hazell, S. L., O'Rourke, J., Kouprach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+spiral-shaped+bacterium+from+the+cat+stomach.">Isolation of a spiral-shaped bacterium from the cat stomach.</a> Infection and Immunity. 56 (11), 2843-2850 (1988).</li><li>Ferrero, R. L., Wilson, J. E., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+Helicobacter-induced+gastric+cancer:+use+of+cocarcinogens.">Mouse models of Helicobacter-induced gastric cancer: use of cocarcinogens.</a> Methods in Molecular Biology. 921, 157-173 (2012).</li><li>Ferrero, R. L., Thiberge, J. M., Huerre, M., Labigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+of+specific-pathogen-free+mice+to+chronic+Helicobacter+pylori+(strain+SS1)+infection.">Immune responses of specific-pathogen-free mice to chronic Helicobacter pylori (strain SS1) infection.</a> Infection and Immunity. 66 (4), 1349-1355 (1998).</li><li>Blanchard, T. G., Nedrud, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Helicobacter+species.">Laboratory maintenance of Helicobacter species.</a> Current Protocols in Microbiology. , (2012).</li><li>Kim, J. S., Chang, J. H., Chung, S. I., Yum, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+host+genetic+background+on+immune+responses+to+Helicobacter+pylori+infection+and+therapeutic+vaccine+efficacy.">Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy.</a> FEMS Immunology and Medical Microbiology. 31 (1), 41-46 (2001).</li><li>Nedrud, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+genetic+influence+on+the+innate+inflammatory+response+to+helicobacter+infection+of+the+gastric+mucosa.">Lack of genetic influence on the innate inflammatory response to helicobacter infection of the gastric mucosa.</a> Frontiers in Immunology. 3, 181 (2012).</li><li>Cai, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+felis+eradication+restores+normal+architecture+and+inhibits+gastric+cancer+progression+in+C57BL/6+mice.">Helicobacter felis eradication restores normal architecture and inhibits gastric cancer progression in C57BL/6 mice.</a> Gastroenterology. 128 (7), 1937-1952 (2005).</li><li>Ferrero, R. L., Labigne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning,+expression+and+sequencing+of+Helicobacter+felis+urease+genes.">Cloning, expression and sequencing of Helicobacter felis urease genes.</a> Molecular Microbiology. 9 (2), 323-333 (1993).</li><li>Stevenson, T. H., Castillo, A., Lucia, L. M., Acuff, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Helicobacter+pylori+in+various+liquid+and+plating+media.">Growth of Helicobacter pylori in various liquid and plating media.</a> Letters in Applied Microbiology. 30 (3), 192-196 (2000).</li><li>Uotani, T., Graham, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+Helicobacter+pylori+using+the+rapid+urease+test.">Diagnosis of Helicobacter pylori using the rapid urease test.</a> Annals of Translational Medicine. 3 (1), 9 (2015).</li><li>Riley, L. K., Franklin, C. L., Hook, R. R., Besch-Williford, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+murine+helicobacters+by+PCR+and+restriction+enzyme+analyses.">Identification of murine helicobacters by PCR and restriction enzyme analyses.</a> Journal of Clinical Microbiology. 34 (4), 942-946 (1996).</li><li>Chaouche-Drider, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+commensal+Helicobacter+sp.+of+the+rodent+intestinal+flora+activates+TLR2+and+NOD1+responses+in+epithelial+cells.">A commensal Helicobacter sp. of the rodent intestinal flora activates TLR2 and NOD1 responses in epithelial cells.</a> PLoS One. 4 (4), e5396 (2009).</li><li>Fox, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicobacter+bilis:+bacterial+provocateur+orchestrates+host+immune+responses+to+commensal+flora+in+a+model+of+inflammatory+bowel+disease.">Helicobacter bilis: bacterial provocateur orchestrates host immune responses to commensal flora in a model of inflammatory bowel disease.</a> Gut. 56 (7), 898-900 (2007).</li><li>McGee, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+enhances+Helicobacter+pylori+resistance+to+antibiotics+and+LL-37.">Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37.</a> Antimicrobial Agents and Chemotherapy. 55 (6), 2897-2904 (2011).</li><li>Viala, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nod1+responds+to+peptidoglycan+delivered+by+the+Helicobacter+pylori+cag+pathogenicity+island.">Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island.</a> Nature Immunology. 5 (11), 1166-1174 (2004).</li><li>Kong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+PCR+method+to+detect+Helicobacter+felis+in+a+conventional+mouse+model.">A sensitive and specific PCR method to detect Helicobacter felis in a conventional mouse model.</a> Clinical and Vaccine Immunology. 3 (1), 73-78 (1996).</li><li>Ng, G., Every, A., McGuckin, M., Sutton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Helicobacter+felis+colonization+in+male+129/Sv+mice+fails+to+suppress+gastritis.">Increased Helicobacter felis colonization in male 129/Sv mice fails to suppress gastritis.</a> Gut Microbes. 2 (6), 358-360 (2011).</li><li>Ferrero, R. L., Ave, P., Radcliff, F. J., Labigne, A., Huerre, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbred+mice+with+long-term+Helicobacter+felis+infection+develop+both+gastric+lymphoid+tissue+and+glandular+hyperplastic+lesions.">Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.</a> The Journal of Pathology. 191 (3), 333-340 (2000).</li></ol>

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...

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。

<table>
	<tbody>
		<tr>
			<td>Bacteriological reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Blood Agar Base No.2</td>
			<td>Thermo Fischer Scientific</td>
			<td>CM0271B</td>
			<td>Dissolve in deinonized water prior to sterilization</td>
		</tr>
		<tr>
			<td>Premium Defibrinated Horse blood</td>
			<td>Australian Ethical Biologicals</td>
			<td>PDHB100</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Brain Heart Infusion Broth</td>
			<td>BD Bioscience</td>
			<td>237500</td>
			<td>Dissolve in deinonized water prior to sterilization</td>
		</tr>
		<tr>
			<td>CampyGen gas packs</td>
			<td>Thermo Fischer Scientific</td>
			<td>CN0035A/CN0025A</td>
			<td></td>
		</tr>
		<tr>
			<td>Histological reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin, neutral buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute alcohol, 100% Denatured</td>
			<td>ChemSupply</td>
			<td>AL048-20L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol (2-propanol)</td>
			<td>Merck</td>
			<td>100995</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene (sulphur free)</td>
			<td>ChemSupply</td>
			<td>XT003-20L</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s Haematoxylin</td>
			<td>Amber Scientific</td>
			<td>&#160;MH-1L</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Eosin, Aqueous Stain</td>
			<td>Amber Scientific</td>
			<td>EOCA-1L</td>
			<td>Filter before use</td>
		</tr>
		<tr>
			<td>Wright-Giemsa Stain, modified</td>
			<td>Sigma Aldrich</td>
			<td>WG80-2.5L</td>
			<td>Dilute before use (20% Giemsa, 80% deionized water)</td>
		</tr>
		<tr>
			<td>Histolene</td>
			<td>Grale Scientific</td>
			<td>11031/5</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>VWR</td>
			<td>1.00579.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligonucleotides</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>The annealing temperature of ureB primers used in this study is 61&#176;C</td>
		</tr>
		<tr>
			<td>GoTaq Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>&#160;M8291</td>
			<td>Kit includes 10X PCR buffer and Magnesium Chloride</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Bioline</td>
			<td>BIO-39028</td>
			<td>Dilute to 10mM in sterile nuclease free water before use</td>
		</tr>
		<tr>
			<td>Molecular Grade Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate</td>
			<td>Univar (Ajax Fine Chemicals)</td>
			<td>A475-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulphate Heptahydrate</td>
			<td>Chem-Supply</td>
			<td>MA048-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Sigma Aldrich</td>
			<td>V2002-1G</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Polymyxin B</td>
			<td>Sigma Aldrich</td>
			<td>P4932-5MU</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Trimethoprim (&#8805;98% HPLC)</td>
			<td>Sigma Aldrich</td>
			<td>T7883</td>
			<td>Dissolve in 100% (absolute) Ethanol</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Amresco (Astral Scientific)</td>
			<td>E437-100MG</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Bacitracin from Bacillus licheniformis</td>
			<td>Sigma Aldrich</td>
			<td>B0125</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Naladixic acid</td>
			<td>Sigma Aldrich</td>
			<td>N8878</td>
			<td>Dissolve in deionized water</td>
		</tr>
		<tr>
			<td>Other reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methoxyflurane (Pentrhox)</td>
			<td>Medical Developments International</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin Wax</td>
			<td>Paraplast Plus, Leica Biosystems</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment and plasticware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Anaerobic Jars</td>
			<td>Thermo Fischer Scientific</td>
			<td>HP0011/HP0031</td>
			<td></td>
		</tr>
		<tr>
			<td>COPAN Pasteur Pipettes</td>
			<td>Interpath Services</td>
			<td>200CS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge</td>
			<td></td>
			<td></td>
			<td>Collect bacterial pellets by centrifugation at 2,200 rpm for 10 mins at 4&#176;C</td>
		</tr>
		<tr>
			<td>23g precision glide needle</td>
			<td>BD Bioscience</td>
			<td>301805</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Bemis, VWR</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Portex fine bore polythene tubing</td>
			<td>Smiths Medical</td>
			<td>800/100/200</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic feeding catheters</td>
			<td>Instech&#160; Laboratories</td>
			<td>FTP20-30</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml tuberculin luer slip disposable syringes</td>
			<td>BD Bioscience</td>
			<td>302100</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf micropestle for 1.2 - 2 mL tubes</td>
			<td>Sigma Aldrich</td>
			<td>Z317314</td>
			<td>Autoclavable polypropylene pestles used for stomach homogenization</td>
		</tr>
		<tr>
			<td>GentleMACs Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>Use a pre-set gentleMACS Programs for mouse stomach tissue</td>
		</tr>
		<tr>
			<td>M Tubes (orange cap)</td>
			<td>Miltenyi Biotec</td>
			<td>30-093-236</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Qubit Fluorometer</td>
			<td>Thermo Fischer Scientific</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic loop</td>
			<td>LabServ</td>
			<td>LBSLP7202</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold Plate, Leica EG1160 Embedding System</td>
			<td>Leica Biosystems</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Base Mould System, Base Mold 38 x 25 x 6</td>
			<td>Sakura, Alphen aan den Rijn</td>
			<td>4124</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek III Uni-Casette System</td>
			<td>Sakura, Alphen aan den Rijn</td>
			<td>4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome, Leica RM2235</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charged SuperFrost Plus glass slides</td>
			<td>Menzel Glaser, Thermo Fischer Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse models of helicobacter infection and gastric pathologies

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。

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</...

Watch this Scientific Journal Video about Mouse Models Of Helicobacter Infection And Gastric Pathologies at JoVE.com

Mouse Models Of Helicobacter Infection And Gastric Pathologies

幽门螺杆菌感染与胃病理的小鼠模型

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。

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

这种方法可以帮助回答胃肠病学领域关于微生物群成员，特别是血小球菌属对炎症和癌症的影响的关键问题。该技术的主要优点是，它允许研究螺旋杆菌感染及其对胃在生理条件下的影响。该技术的影响延伸到开发用于预防或消除螺旋杆菌感染及其后果的疗法，包括抗生素、药物或疫苗。这些方法提供对螺旋杆菌感染的发病机制的见解。然而，一些技术可能适应其他胃肠道病原体的研究。一般来说，新方法的个体将难以制备能够建立小鼠感染的细菌注射剂。Helicoli 细菌分离物是已知的小鼠殖民菌株，这些菌株在体外没有经过广泛的亚培养。此外，应记录这些菌株的体外亚栽培数量。将细菌悬浮液加入 15 毫升聚苯乙烯管中，并使用塑料环将每个培养的 10 到 20 微升液滴转移到单独的玻璃显微镜幻灯片上。然后，以100倍的放大率评估相对比显微镜下细菌的生存能力和活力。只有当大多数细菌具有杆状时，才使用 H.pylori 接种。H.felis接种应主要含有利形细菌。将接种器装入单独的一次性单毫升注射器中。为每个注射器配备一个 23 量针，并使用塑料石蜡膜将一次性聚乙烯导管连接到每根针上。接下来，通过颈部和尾部的擦伤，手动约束一只六到八周大的特异性无病原体和无螺旋杆菌的小鼠，并将一个导管插入打开的下颚中心。引导导管在导管向食道，延长小鼠的脖子，让轻松通过食道进入胃和远离气管，直到大多数或所有的导管不再可见，并感觉到阻力。然后，将至少10倍的细菌送到第五细菌，以确保最佳的殖民化和疾病病理学。实验结束时，用细的弯曲剪刀打开腹腔切除胃部。沿着更大的曲率切开胃，轻轻洗两次器官，每次洗涤一个50毫升的新鲜PBS管，以去除任何残留的食物。第二次洗涤后，将组织放入焦油、六厘米的塑料培养皿中，以记录湿重。平整胃部后，将样品分分两部分，分成两个相等的组织片段，包括蚂蚁、身体和非腺膜区域。取出非腺体区域，称重每个胃的一半，然后再将一块组织储存在适当的溶液中，用于计划的下游分析。将胃组织的另一半加入含有10%甲醛的15毫升圆锥管中，10秒后将组织压扁在管顶面。当组织被贴在管壁上时，将样品重新浸入甲醛中至少24小时。计算感染后胃中可行的H.pylori的数量，使储存在BHI中的胃样本均质化，并在新鲜、无菌的汤中对产生的胃同质质进行重复的连续稀释。将预干马血加量（HBA）的盘子，加上额外的抗生素，分成三或四个部分，并使用 Miles 和 Misra 技术的适应，在每个胃的一段上添加 10 到 100 微升均质稀释剂。用无菌的塑料环铺开同质物，让板干燥。然后，将盘子倒置在含有培养皿水和气体包装的气罐中。然后，在37摄氏度下孵化罐子4至7天。当可以观察到菌落时，枚举包含 10 到 100 个孤立菌落的段。安乐死后，胃从动物身上收获并称重。非腺区被移除，胃被分成两个相等的一半，包括蚂蚁和身体。成功的殖民化通常通过执行可行的计数和随后从条纹到HBA板的胃同质物中对单个菌落进行计数来确认。请注意，从小鼠胃微生物群或大量H.pylori菌落中感染细菌的存在可能会使H.pyloriCFO的枚举复杂化。或者，PCR 可用于使用针对 H.felis 和 H.pylori ureB 基因的 325 基对区域的特定验证底向器来验证感染。在这个具有代表性的实验中，野生型C57 Black-6小鼠表现出中度炎症迹象，包括感染H.felis后六个月的增生和腺体萎缩，以及亚木科萨的细胞渗透。在同一时间点，在敲除小鼠中观察到更严重的炎症，在靠近细胞渗透的地方观察到淋巴卵泡的附加存在。H.felis 细菌也可以在受感染的小鼠胃组织的吉姆萨染色部分可视化。按照这个程序，可以执行其他方法，如定量PCR，免疫组织化学，或免疫荧光，以回答有关在螺旋杆菌感染期间诱导的宿主反应类型的其他问题。该技术开发后，为微生物学和胃肠病学领域的研究人员探索幽门螺杆菌在人类胃病中的因果关系铺平了道路。看完这段视频后，你应该对如何复制感染与螺旋杆菌物种的小鼠有一个良好的理解，并研究与感染相关的病理学。不要忘记，本协议中使用的 Helico 细菌菌株具有传染性，因此在使用这些细菌时应始终遵循标准的生物安全程序。

Research

JoVE Journal

Immunology and Infection

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Transurethral Induction of Mouse Urinary Tract Infection

经尿道诱导小鼠尿路感染

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

科研

免疫与感染

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Ecto和实验室大鼠和小鼠体内寄生虫的诊断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

类器官的模型传染病：人类的文化与小鼠胃组织体与幽门螺杆菌的显微注射

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

的气管内管理<em&gt;流感嗜血杆菌</em&gt;在小鼠模型来研究气道炎症

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

小鼠和人源性胃上皮单层培养对再生的研究

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

颅内 Subarachnoidal 感染对猪链球菌生物膜在小鼠感染模型中作用的研究

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

基于 crispr-cas9 的基因组工程, 用于生成 hiv-1 感染与选定的证明病毒整合点的 jurkat 报告模型

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

人类免疫缺陷小鼠模型中的慢性、急性和再激活HIV感染

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...