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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+long-term+cervical+persistence+of+Chlamydia+trachomatis+by+omp1+genotyping.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Chlamydia+trachomatis+deoxyribonucleic+acid+in+women+with+tubal+infertility.">Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility.</a> Fertil. Steril. 59, 45-50 (1993).</li><li>Peipert, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Genital+chlamydial+infections.">Clinical practice. Genital chlamydial infections.</a> N. Engl. J. Med. 349, 2424-2430 (2003).</li><li>Mardh, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubal+factor+infertility,+with+special+regard+to+chlamydial+salpingitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway-specific+differences+between+tumor+cell+lines+and+normal+and+tumor+tissue+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+gamma+interferon-mediated+antichlamydial+defense+mechanisms+in+human+and+mouse+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+mice+and+not+men:+differences+between+mouse+and+human+immunology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+is+the+initiator+of+Fallopian+tube+destruction+during+Chlamydia+trachomatis+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+abrogates+antichlamydial+properties+of+IFN-gamma+in+human+fallopian+tube+cells+in+vitro+and+ex+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-exposure+of+infected+human+endometrial+epithelial+cells+to+penicillin+in+vitro+renders+Chlamydia+trachomatis+refractory+to+azithromycin.">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><li>Van Voorhis, W. C., Barrett, L. K., Sweeney, Y. T., Kuo, C. C., Patton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Chlamydia+trachomatis+infection+of+Macaca+nemestrina+fallopian+tubes+produces+a+Th1-like+cytokine+response+associated+with+fibrosis+and+scarring.">Repeated Chlamydia trachomatis infection of Macaca nemestrina fallopian tubes produces a Th1-like cytokine response associated with fibrosis and scarring.</a> Infect. Immun. 65, 2175-2182 (1997).</li></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

12.8K 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

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

科研

医学

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

伤口愈合的小鼠模型

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

小鼠加速动脉硬化的诱导："电线损伤"模型

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

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 ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...