这项工作是由东风集团群集卓越“炎症接口”（RA，如果RA - D）支持。我们为克里斯汀Wischnat优秀的技术援助表示感谢。

1。 沙眼衣原体血清型D投入的制备<ol><li>感染合流175厘米2细胞培养瓶摄氏用4毫升含有的HeLa-229细胞衣原体 D在石药缓冲区。 </li><li>在不断晃动在37°C孵育一小时。 </li><li>添加10％胎牛血清和240μL，放线菌酮20毫升DMEM培养液。 </li><li>在加湿孵化器的孵育48小时37°C间和5％的CO 2。 </li><li>加入5毫升无菌玻璃珠，强烈的震动，分离和破坏C。沙眼窝藏的HeLa-229细胞。 </li><li>收集上清，添加另一个无菌玻璃珠的5毫升。 </li><li>岩3次为1分钟，以销毁所有的HeLa-229细胞，并释放C。沙眼 。 </li><li>离心机在1000 RPMI 5分钟和4°C至去除细胞碎片。 </li><li>收集8日至10 175厘米2细胞培养上清感染瓶合流Ğrown与HeLa细胞229细胞。 </li><li>每一瓶孵育4毫升石药缓冲区和4毫升从以前的感染上清一小时在37°Ç不断晃动。 </li><li>一个小时后，添加10％胎牛血清和230μL，放线菌酮在37°C和5％CO 2孵育48小时15毫升培养液。 </li><li>加入5毫升无菌玻璃珠，摇来分离和破坏C。沙眼窝藏的HeLa-229细胞。 </li><li>收集上清，添加另一个无菌玻璃珠的5毫升。 </li><li>岩3次为1分钟，以销毁所有的HeLa-229细胞，并释放C。沙眼 。 </li><li>在1000 RPMI 5分钟的离心机在4°C，以去除细胞碎片。 </li><li>收集上清。 </li><li>填写在50毫升的猎鹰管，离心99分钟11000 RPMI 40上清毫升。 </li><li>离心后弃上清，用1毫升盛高置缓冲区洗沉淀。 </li><li>均质的颗粒抢断和1毫升，分装20μL1，5毫升Eppendorf管中，储存于-70℃直至使用Ç。准备股票的生物活性被证实在一个既定的HeLa-229细胞感染模型。 </li></ol> 2。人输卵管的制备<ol><li>商店人输卵管（HFT）的，在无抗生素的RPMI含5％FCS在4°C间，直到准备手术后立即接受子宫切除术的妇女。吕贝克大学（09-153）伦理委员会批准该协议。包括在研究中（34至53岁）的妇女没有之前三历史衣原体感染。输卵管组织，剖腹产对产妇的要求，在收集期间在黄体期症状性子宫肌瘤，由于子宫切除在围产期杀菌。所有的哈沃被检测呈阴性的 C语言衣原体的PCR。 </li><li>剖析在一个Petri菜含有的RPMI与哈沃5％胎牛血清无抗生素。在整个处理的组织应该被淹没在媒体，以防止干燥。 </li><li>切断并丢弃在手术过程中破坏结缔组织和组织。 </li><li>哈沃仔细解剖后打开一个小手术刀。 </li><li>为进一步的实验准备在0.5厘米×0.5厘米至1厘米×1厘米大小的组织块。 </li><li>感染商店哈沃标本1毫升不含抗生素的RPMI含有5％胎牛血清。哈沃标本5.5x10 5 IFU的形成单位）（包含C 的感染沙眼 。 </li><li>哈沃标本孵育在37℃，5％CO 2和O 2的 21％以及在37℃，5％CO 2和2％的O 2 24小时。 </li><li>孵化时间后收集蒙蒂的固定液的标本和存储在4°C，直到扫描电镜/ TEM准备至少3天。 </li></ol> 3。透射电镜哈沃试样的制备<ol><li>为透射电镜从蒙蒂的固定液的标本，并削减2×2毫米到4×5毫米件。其余组织可用于扫描电镜。 </li><li>洗件二甲砷酸钠缓冲液，pH值7.35 1小时。 </li><li>孵化在1％（W / V）旱厕目的的四氧化锇。过夜。 </li><li>二甲砷酸钠缓冲液，pH值7.35洗6X5分钟，取出多余的四氧化锇。 </li><li>删除在水，乙醇（V / V）浓度的增加（2小时30％，40％为2小时，2小时50％，60％为2小时，晚上超过70％，80％为2孵化水H，90％，95％为1小时1小时，1小时的100％。 </li><li>洗出乙醇和环氧丙烷为2×15分钟后，转移到新鲜配制的丙烯（包括所有组件）牢50％的混合物（V / V），并孵育过夜。 </li><li>转拨至新鲜制备的牢（包括所有组件），至少1小时。 </li><li>转移到平坦嵌入模具，并填写与牢。 </li><li>让为牢变硬至少48小时在60°C </li><li>嵌入式样本修整后切半薄超薄切片的路段上使用玻璃刀或的直方图钻石刀（700毫微米），并与理查森的污点污点。 </li><li>切超薄切片（约70纳米）使用钻石刀和铜网格（如150平方米的网）转移到。 </li><li> 0.5％（W / V）在旱厕目的的铀醋酸染色超薄切片。其次为3％（W / V）铅柠檬酸在Aqua目的。自动节stainer。另外，部分可染色手动离开电网铀在旱厕目的醋酸离心饱和溶液15分钟。其次是0.3％柠檬酸铅在旱厕目的（W / V）。 </li></ol> 4。哈沃试样的制备扫描电镜<ol><li>扫描电镜从蒙蒂的固定液，东方标本1.5×1厘米软木与上皮面临向上，用昆虫针固定位置。 </li><li>软木标本转移到合适的金属筐和洗为30分钟二甲砷酸钠缓冲液，pH值7.35。 </li><li>删除由孵化试样在丙酮/水混合物的浓度（V / V）30％，为6小时，6小时40％，60％为8小时，晚上超过70％，80％为2小时，90％的水2 h和100％以上夜间极为谨慎，以保持标本不断淹没。 </li><li>临界点干燥（温度40℃，压力80巴），转移到新鲜的100％丙酮和干燥标本。 </li><li>删除面临的上皮向上SEM试样安装配备使用导电碳水泥标签用导电碳针，软木和胶水标本。使用液体的导电银之间的安装和导电碳标签，以提高电导率。 </li><li>准备一个用溅射镀膜机试样的导电铂金，黄金或钯涂层。 </li></ol> 5。理查森的色漆半薄切片染色<ol><li>让半薄片干幻灯片的。 </li><li>染色染色1-2分钟的解决方案，在60°C的部分。 </li><li>洗目的旱厕。 </li><li>干节和盖玻片。 </li></ol> 6。代表结果在人类的输卵管感染模型，我们能够想象在上皮细胞形态的差异非感染对照组（ 图1）和C。沙眼 -常氧条件下的感染管（ 图2）。形态特征差异暗示病原体引起的，不能在非感染控制检测输卵管上皮细胞肿胀和溶解。采用半薄切片和透射电子显微镜，我们能够证明细胞内三衣原体体外感染人输卵管组织中的包裹体形成（ 图3，5），但在非感染控制（ 图4）。由于氧浓度在炎症过程中，我们还调查了我们的模型，在缺氧条件下，在女性生殖道的的tions已经在生理条件下11，并进一步减少低。初步结果表明，该模型是有用的分析衣原体的生长和后代在不同氧气浓度。衣原体感染引起的上皮细胞损伤，必须通过不谨慎的准备和处理输卵管感染前（ 图6）介导的文物分离。 <img src="/files/ftp_upload/4036/4036fig1.jpg" alt="图1" /> 图1。控制介质的一天后，潜伏期（绿色箭头显示纤毛细胞，蓝色箭头显示非纤毛细胞）的扫描电子显微镜的非感染人类输卵管（SEM）。 <img src="/files/ftp_upload/4036/4036fig2.jpg" alt="图2" /> 图2。扫描电子显微镜<html>副本（SEM）的体外C.衣原体感染人类输卵管天感染后（白色箭头标记破裂列入）。 <img src="/files/ftp_upload/4036/4036fig3.jpg" alt="图3" /> 图3。半薄切片显示与 C人类输卵管感染后的典型内夹杂物（白色箭头）1D 衣原体体外 。 <img src="/files/ftp_upload/4036/4036fig4.jpg" alt="图4" /> 图4。非感染人类输卵管天孵化后，在控制介质的透射电子显微镜（TEM）。 <img src="/files/ftp_upload/4036/4036fig5.jpg" alt="图5" /> 图5。透射电子显微镜（TEM）的体外C.衣原体感染人类输卵管天感染后（白色箭头标记衣原体包裹）。 ve_content“&gt; <img src="/files/ftp_upload/4036/4036fig6.jpg" alt="图6" /> 图6。扫描电子显微镜（SEM）输卵管上皮通过不谨慎试样的制备和处理（白色箭头标记破坏上皮组织）破坏。

<ol><li>Dean, D., Suchland, R. J., Stamm, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+long-term+cervical+persistence+of+Chlamydia+trachomatis+by+omp1+genotyping%5BTitle%5D)+AND+%22J.+Infect.+Dis%22%5BJournal%5D)">Evidence for long-term cervical persistence of Chlamydia trachomatis by omp1 genotyping.</a> J. Infect. Dis. 182, 909-916 (2000).</li>
<li>Campbell, L. A., Patton, D. L., Moore, D. E., Cappuccio, A. L., Mueller, B. A., Wang, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+Chlamydia+trachomatis+deoxyribonucleic+acid+in+women+with+tubal+infertility%5BTitle%5D)+AND+%22Fertil.+Steril%22%5BJournal%5D)">Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility.</a> Fertil. Steril. 59, 45-50 (1993).</li>
<li>Peipert, J. F. Clinical practice. Genital chlamydial infections. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aPeipert%2C+JF+%22N.+Engl.+J.+Med%22">N. Engl. J. Med</a>. 349, 2424-2430 (2003).</li>
<li>Mardh, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tubal+factor+infertility%2C+with+special+regard+to+chlamydial+salpingitis%5BTitle%5D)+AND+%22Curr.+Opin.+Infect.+Dis%22%5BJournal%5D)">Tubal factor infertility, with special regard to chlamydial salpingitis.</a> Curr. Opin. Infect. Dis. 17, 49-52 (2004).</li>
<li>Ertel, A., Verghese, A., Byers, S. W., Ochs, M., Tozeren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathway-specific+differences+between+tumor+cell+lines+and+normal+and+tumor+tissue+cells%5BTitle%5D)+AND+%22Mol.+Cancer%22%5BJournal%5D)">Pathway-specific differences between tumor cell lines and normal and tumor tissue cells.</a> Mol. Cancer. 5, 55(2006).</li>
<li>Roshick, C., Wood, H., Caldwell, H. D., McClarty, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+gamma+interferon-mediated+antichlamydial+defense+mechanisms+in+human+and+mouse+cells%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells.</a> Infect. Immun. 74, 225-238 (2006).</li>
<li>Mestas, J., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+mice+and+not+men%3A+differences+between+mouse+and+human+immunology%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Of mice and not men: differences between mouse and human immunology.</a> J. Immunol. 172, 2731-2738 (2004).</li>
<li>Hvid, M., Baczynska, A., Deleuran, B., Fedder, J., Knudsen, H. J., Christiansen, G., Birkelund, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-1+is+the+initiator+of+Fallopian+tube+destruction+during+Chlamydia+trachomatis+infection%5BTitle%5D)+AND+%22Cell+Microbiol%22%5BJournal%5D)">Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection.</a> Cell Microbiol. 9, 2795-2803 (2007).</li>
<li>Roth, A., Konig, P., van, Z. G., Klinger, M., Hellwig-Burgel, T., Daubener, W., Bohlmann, M. K., Rupp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypoxia+abrogates+antichlamydial+properties+of+IFN-gamma+in+human+fallopian+tube+cells+in+vitro+and+ex+vivo%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Hypoxia abrogates antichlamydial properties of IFN-gamma in human fallopian tube cells in vitro and ex vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 19502-19507 (2010).</li>
<li>Wyrick, P. B., Knight, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pre-exposure+of+infected+human+endometrial+epithelial+cells+to+penicillin+in+vitro+renders+Chlamydia+trachomatis+refractory+to+azithromycin%5BTitle%5D)+AND+%22J.+Antimicrob.+Chemother%22%5BJournal%5D)">Pre-exposure of infected human endometrial epithelial cells to penicillin in vitro renders Chlamydia trachomatis refractory to azithromycin.</a> J. Antimicrob. Chemother. 54, 79-85 (2004).</li>
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</ol>

没有利益冲突的声明。

危害病原体引起的感染组织的可视化是艰巨的，往往是有限的，在小鼠实验程序。我们建立在人输卵管体外感染模型来分析C。上的女性生殖道衣原体感染。通过使用这种方法，我们能够可视化C。沙眼 -人输卵管上皮细胞损伤，感染后一天之内。细胞肿胀和溶解是典型的形态学变化， 在 C 沙眼 -输卵管感染，但在非感染控制。 TEM分析表明，衣原体在受感染的组织实施的一个典型的细胞内发育周期，显示重新分化的基本机构，而且还扩大网纹可能表明持久的机构小包裹大包裹。然而，单独的形态图片没有足以区分REPL的icating和持续感染，减少代谢特点和增加耐抗菌素10。 在受感染的输卵管上皮细胞的破坏，要么是由于C 的中断沙眼 -完成后的细胞发育周期和传染病的基本机构释放或释放促炎细胞因子8感染的上皮细胞。病原体引起的组织损伤和对C.主机诱导的免疫反应衣原体被认为是导致上皮细胞的功能，重塑成纤维细胞在体内 8,11终于输卵管不孕的损失的主要因素。 这种模式的主要限制之一是炎性细胞的情况下，这阻碍了初始C.辅助疗效分析衣原体感染输卵管上皮细胞。然而，该模型是APpropriate探讨不同环境条件（如氧含量）和最初的宿主免疫反应在急性三衣原体感染8,9。进一步的计划是建立一个测试模型对三抗菌素沙眼在整个人类的输卵管，在输卵管上皮细胞双光子激光扫描显微镜观察不同发育阶段的特征的代谢状态。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液</td><td>临时机场管理局</td><td> E15-843 </td><td></td></tr><tr><td>的RPMI </td><td>临时机场管理局</td><td> R15-8​​02 </td><td></td></tr><tr><td> FCS的</td><td>临时机场管理局</td><td> A15-101 </td><td></td></tr><tr><td>放线菌酮</td><td> Sigma - Aldrich公司</td><td></td><td></td></tr><tr><td>石药缓冲区</td><td>各个</td><td></td><td>看到配方如下</td></tr><tr><td>蒙蒂的固定液</td><td>各个</td><td></td><td>看到配方如下</td></tr><tr><td>二甲砷酸钠缓冲</td><td>各个</td><td></td><td>看到配方如下</td></tr><tr><td> ŕichardson的污点</td><td>各个</td><td></td><td>看到配方如下</td></tr></tbody></table>食谱： 1。石药缓冲区<ol><li>加入75 g蔗糖。 </li><li>添加2.47克磷酸二钠。 </li><li>添加0.36克磷酸二氢钠。 </li><li>添加0.72克谷氨酸。 </li><li>填充到1 L水作空白。 </li><li>调节pH值至7.3。 </li></ol> 2。 0.1米二甲砷酸钠缓冲液，pH值7.35。 <ol><li>添加42.80克二甲基胂酸的钠盐。 </li><li>添加蔗糖68.46克。 </li><li>填充到1 L水作空白。 0.2M的二甲砷酸钠缓冲。 </li><li>前使用旱厕目的的稀溶液。到所需的浓度，并调整pH值至7.35，如果必要。 </li></ol> 3。蒙蒂的固定液<ol><li>加入155毫升0.12 M二甲砷酸钠缓冲。 3.2在旱厕目的添加25毫升glutardialdehyde 25％。 </li><li>添加19毫升“10％多聚甲醛在Aqua目的。 </li><li>加入3毫升3％，在旱厕目的氯化钙2。 </li><li>调整pH值至7.35和填补与旱厕的目的。 312毫升的最终解决。 </li></ol> 4。理查森的污点<ol><li> 1％亚甲蓝混合在1％水溶液的硼砂溶液和1％的水作空白，海岸第二等份。 </li></ol>

a human fallopian tube model for investigation of c. trachomatis infections

沙眼衣原体生殖道感染（ 沙眼衣原体 ）是最常见的性传播疾病在世界各地的妇女。低效的间隙或病原体的持久性，可能会导致升上生殖道感染，应该引起1,2感染组织的慢性炎症损害。因此，像盆腔炎（PID）的，输卵管阻塞，不孕不育的临床后遗症严重，可能会出现3,4。 与 C的研究沙眼已在上皮细胞株（如HEp-2细胞和HeLa-229）或小鼠进行。然而，由于与细胞培养模型，他们既不反映本地组织的生理，也不是C 的病理生理在体内 5 衣原体生殖道感染。进一步限制的事实，中央信号级联（如干扰素γmediat的编辑JAK / STAT信号通路），控制细胞内衣原体的增长从根本上老鼠和人类6,7之间的差异。我们和其他人，因此建立一个整体的器官输卵管模型，探讨之间C.直接的相互作用衣原体和人类输卵管细胞体外 8,9。 为了这个目的，人类从接受子宫切除术的妇女输卵管收集和感染与 C 衣原体血清型D.感染后24小时之内，样品分析使用扫描电子显微镜（SEM）和透射电子显微镜（TEM）检测沙眼衣原体介导的上皮损伤，以及C.衣原体在输卵管组织列入的形成。

Watch this Scientific Journal Video about A Human Fallopian Tube Model for Investigation of C. trachomatis Infections at JoVE.com

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

我们描述了一个<em&gt;体外感染</em&gt;直接从细菌病原体与人类输卵管细胞相互作用的可视化模型。整个器官组织模式成立调查<em&gt; C。沙眼</em&gt;引起的病理条件下的“生命”的女性输卵管。

一个人的调查输卵管模型<em&gt; C。沙眼</em&gt;感染

Genital tract infections with Chlamydia trachomatis (C. trachomatis) are the most frequent transmitted sexually disease in women worldwide. Inefficient ...

a-human-fallopian-tube-model-for-investigation-c-trachomatis

Research

JoVE Journal

Medicine

A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

12.9K 次观看.  University of Lübeck. 我们描述了一个体外感染直接从细菌病原体与人类输卵管细胞相互作用的可视化模型。整个器官组织模式成立调查 C。沙眼引起的病理条件下的“生命”的女性输卵管。

视频: A Human Fallopian Tube Model for Investigation of C. trachomatis Infections

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

鞭虫感染muris：在肠道内的2型免疫和炎症模型

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

科研

免疫与感染

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小动物模型尿路感染的实验研究: 男性和女性的经尿道插管

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

An Efficient Method for Extracting Human Fallopian Tube Epithelia for Single-cell Analyses

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if fertilization is successful, the embryo. The epithelial cells that line the inner surface of fallopian tubes are integral to both normal and abnormal fallopian tube processes, including disease initiation. After menopause, fallopian tubes cease to have a significant role in the body and the intraepithelial cell makeup changes. We describe a method in which these epithelial cells can be isolated from fresh fallopian tubes with minimal stromal cell contamination into a single-cell suspension. These cells can be grown in culture or used for further analysis such as flow cytometry and single-cell RNA sequencing. This isolation protocol can be achieved in 4-6 h and yields viable cells that can be used for immediate downstream analysis. This efficient protocol facilitates the isolation of fallopian tube epithelial cells with an enriched epithelial population.

This protocol describes a one-day method for the isolation of human fallopian tube epithelial cells. Isolated epithelial cells can be plated in 2-dimensional (2D) culture or dissociated into single-cell suspensions and utilized in downstream experiments, including flow cytometry and single-cell RNA sequencing.

提取人输卵管上皮细胞用于单细胞分析的有效方法

Human fallopian tubes are intrinsic to reproduction. The purpose of the fallopian tubes is to allow for the transit of sperm, the ovum, and if ...

癌症研究

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

生物学

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

输卵管的管状细胞学作为卵巢癌早期检测的有前景的工具

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

医学

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as coccidiosis and diet, with gavage or administration via the feed using Clostridium perfringens. In these models, the comparison of multiple C. perfringens strains for virulence studies requires a large number of hosts to obtain significant results. Mortality during the course of the study can be high depending on the experimental model, hence raising ethical concerns regarding animal welfare in research. The development of new infection models requiring fewer animals to study pathogenesis, yet providing statistically significant and valid results, is important in reducing animal use in research. Intestinal ligated loop models have been used to study clostridial infections in various species such as mice, rabbits and calves. Following surgical procedures to create ligated loop segments, C. perfringens strains are injected directly into the loops to establish a close contact between the bacteria and the intestinal mucosa. Samples of the small intestine and luminal contents are taken at the termination of the procedures after a few hours. Multiple bacterial strains can be inoculated in each animal, hence reducing the number of required subjects in the experiments. Also, procedures are performed under general anesthesia to reduce animal pain. In chickens, this model would be more appropriate than oral administration to compare C. perfringens strain pathogenicity because fewer animals are needed, no predisposing factors are required to induce the disease, and pain is controlled by analgesics. The intestinal ligated loop model is poorly described in chickens and standardization is essential for its optimal use. This manuscript provides all the necessary steps to create numerous intestinal ligated loops in chickens and brings information on the critical points to obtain valid results.

A Ligated Intestinal Loop Model in Anesthetized Specific Pathogen Free Chickens to Study Clostridium Perfringens Virulence

Here we present a protocol to surgically create &#39;intestinal ligated loops&#39; in chicken small intestines. This procedure allows for the comparison of multiple Clostridium perfringens strains&#39; virulence in situ in a single host. This method markedly decreases the number of chickens usually necessary for similar in vivo experiments.

麻醉特异性病原体游离鸡结扎肠环模型研究荚梭菌毒力

Necrotic enteritis was studied in chickens using various in vivo infection models. Most of these use a combination of predisposing factors, such as ...

Comprehensive Approach for Microbial Isolation from Hidradenitis Suppurativa Tunnels

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's dermal layers, causing significant discomfort, foul-smelling discharge, disfigurement, contractures, and scarring, which severely diminish the quality of life. HS is associated with alterations in the skin microbiome, impacting immune regulation and the skin's defense against harmful bacteria. Despite its prevalence, the contribution of the HS microbiome to disease pathology and the limited response to treatment remains largely unknown. To date, multiple 16S rRNA sequencing studies on HS tissue have only achieved genus-level granularity, identifying an increase in Gram-negative anaerobes and a decrease in skin commensals. A deeper understanding of microbial dysbiosis in individuals with HS is essential for optimizing treatment strategies. This requires a two-pronged approach to assessing the HS microbiome, including the isolation of bacterial species, which are often underutilized in translational studies focused on skin disorders. Isolating individual microorganisms from HS tissue is crucial for elucidating the role of bacteria in HS pathogenesis. Here, we highlight reproducible methods to successfully isolate anaerobic pathogens from HS tunnel tissue, providing the initial and most critical step in understanding bacterial role in HS. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address the complex microbial burden of this chronic condition.

We showcase a method to successfully isolate fastidious and anaerobic organisms from cutaneous sinus tracts (tunnels) in tissues excised from patients with Hidradenitis Suppurativa.

从化脓性汗腺炎隧道中分离微生物的综合方法

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's ...

Papanicolaou Staining: A Technique to Visualize the Cytological Features of Tubal Cells

Source: Chen, H. et al. <a href="https://www.jove.com/v/55887">Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection.</a> J. Vis. Exp. (2017).
In this video, we demonstrate a modified protocol of Papanicolaou staining to study tubal cytology. This technique can be used to visualize the cytological features of malignant and non-malignant fallopian tube epithelial cells.

巴氏染色:一种可视化输卵管细胞细胞学特征的技术

Source: Chen, H. et al. Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection. J. Vis. Exp. (2017).
In this video, ...

JoVE Encyclopedia of Experiments

Biological Techniques

Staining Techniques

Cell staining

Transurethral Catheterization and Bacterial Inoculation to Generate a Urinary Tract Infection Model in Male Mice

Source: Zychlinsky Scharff, A., et al. <a href="https://app.jove.com/v/54432">Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.</a> J. Vis. Exp. (2017).
This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

经尿道导管插入术和细菌接种在雄性小鼠中生成尿路感染模型

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunology

Immunopathology

Host-Pathogen Interaction Models

Establishment of a Urinary Tract Infection Model in Female Mice

Source: Zychlinsky Scharff, A. et al., <a href="https://app.jove.com/v/54432">Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the establishment of uropathogenic Escherichia coli infection in a female mouse bladder through transurethral catheter instillation. The injected bacteria successfully establish a urinary tract infection within the bladder, providing a valuable platform for studying healthcare-associated diseases related to the urinary tract.

一个人的调查输卵管模型

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