这项工作是由科学技术部资助项目（MOST 103-2633-B-002-002; 104-2320-B-002-048）。我们感谢博士。贝蒂吴和谢建库·李在台大，利·泽博尼在斯坦福大学和彼得·奥夫曼博士在夏威夷大学读书的手稿，尤恩·奇恩台大技术援助，以及文奇·韦博士在农业生物技术研究中心中央研究院的技术咨询。

在8只雌性小鼠 - 12周龄被用于这项研究。 C57 / BL6野生型（WT）和IL-15缺陷（IL15 - / - ）小鼠分别从国家实验动物中心（NLAC），台湾和Taconic的农场购买。 IL-15缺陷型（IL15 - / - ）和IL-15-显性（IL15 +/-和IL15 + / +）显示出1:3的比例在第二代从IL15 +/-杂合子的横。 IL-15的基因型- / -小鼠通过PCR分析证实。小鼠保持在实验动物中心（LAC），国立台湾大学医学院（NTU）学院无特定病原（SPF）设施。所有的动物程序，包括麻醉材料和方法按照由医学国立台湾大学和学院公共卫生机构动物护理和使用委员会（IACUC）（动物协议20130225批准的证明）批准的动物方案进行。 &lt;P类= "jove_title"&gt; 1。针灸针的制备注:针灸针是一手柄和一针端（ 图1A）构成的医疗器械。针刺针的直径为0.2毫米（36 G）至0.35毫米（28 G）范围内。我们选择针的最好的点，以避免刺皮肤期间可能出现的过度的细胞损伤。有4个不同的0.2毫米的针的长度包括13毫米（0.5英寸），25毫米（1.0英寸），40毫米（1.5英寸），并在长度为50毫米（2.0英寸）。的10针13毫米长的bundle上C57BL / 6小鼠皮肤振荡针期间提供最佳舒适的手柄。如果该方法适用于较大的动物的皮肤或由研究人员巨掌执行的参数可以修改。 <ol><li>从无菌的，无热原的包装中取出针灸针。将针在无菌铺巾。 </li><li>使用粘合剂标签带10针结合我一起NA束（ 图1B）。 注:10针几何方式布置，以形成圆柱状。为了方便起见，使用一块石蜡膜的涂敷粘合带之前稳定布置。确保所有的针点在同一平面上。使用束而不是单个针通过增加针和皮肤之间的接触面积有利于最大DNA输注。 </li></ol> 2.大量的制备和无内毒素的质粒DNA <ol><li>变换用质粒DNA化学感受细菌细胞。 </li><li>选择并在3mL LB的含有抗生素的培养基接种单一菌落，并在摇床中在37℃下孵育过夜。 </li><li>用抗生素100在250ml的LB培养基中并在37℃振荡培养过夜C.:通过在1稀释细菌培养物扩大培养</li><li>通过使用高qualit制备DNA的大量Y，按照制造商的说明书，无内毒素的质粒制备试剂盒。 注:Acufection协议需要高纯度和质粒DNA的量大。高质量的使用，无内毒素和柱纯化的质粒DNA的建议。 </li><li>洗脱在无菌水中DNA。商店DNA以小等分试样在-20℃下直至准备acufection。 注意:避免DNA的反复冻结与解冻，以确保一致的质粒DNA的表达。 </li><li>稀释质粒DNA至1毫克/毫升在无菌PBS中进行acufection。 </li></ol> 3.程序Acufection 注:DNA的最佳量和目标皮肤表面的面积可以针对不同的基因变化，需要进一步优化。穿刺力和次数松开皮肤的角质层也可以根据皮肤的厚度而变化。这些条件必须评估acufected格涅·特兰斯克里的表达水平后小心地确定PTS通过qRT-PCR。 <ol><li>称量小鼠和给予腹腔注射三溴乙醇（每克体重400微克）。 注:2,2,2-三溴是被普遍用于小鼠注射的麻醉剂。溶液是通过溶解在含有2-甲基-2-丁醇的2.5％的蒸馏水（200mL）中的非医药级2,2,2-三溴乙醇（2.5克）制备。 </li><li>使用在眼睛兽医眼科软膏以防止麻醉下干燥。 注: - 2分钟麻醉作用将在1来诱导。检查脚趾捏反射，以保证手术麻醉前的足够的深度。 </li><li>剃须脊侧皮肤，适用脱毛霜，以溶解角质蛋白在毛干。 注:使用的脱毛霜去除头发将促进DNA的注射入皮肤。 </li><li>用清水浸泡棉球擦去脱毛膏。 注:脱毛膏是易溶于水。彻底清除霜西港岛线升防止油腻的表面，并确保更好的刺结果。 </li><li>使用70％的乙醇浸泡消毒棉签消毒皮肤表面。 </li><li>标记目标面积（1cm×1cm的）与预先测定的模版的脱毛皮肤。注:标记目标站点，将有助于应用针束向上和向下的限定区域内。这是重要的，以确保相同量的DNA递送至的比表面积，以尽量减少实验对实验变异。 </li><li>放置质粒DNA（10微克）的到显着皮肤上的10μL降。 注:DNA为acufection的量随不同的基因和表达载体的类型而变化。使用DNA的量应该进行感兴趣的实验前应仔细滴定。 10μL是小液滴和它位于井的剃和脱掉的皮肤无滚下而穿刺。包括用10μL空载体DNA处理的小鼠的对照组小鼠比较cufected与研究的基因。 </li><li>握住针刺针束（ 图1B），并用100次，或直到从DNA在PBS中的水分在皮肤上消失的向上和向下运动刺破标记表面。注:可能出现皮肤表面的有些发红。不要制定那些流血大幅削减。当从DNA在PBS中的水分（10μL）在皮肤上消失可操作地确定向上和向下的在皮肤表面上穿刺运动的数量。我们在30秒决定100倍，因为它给了从acufection交付基因最一致的结果。如果适用，该针可重复使用多达6个目标皮肤刺（1 1cm×1cm的每个）。冲洗在70％乙醇和PBS acufection之间的针。更改为新的针时它们索然无味或不同的质粒DNA，以避免交叉污染。 </li><li>放置在加热垫上acufected小鼠以维持体温，直到清醒。 </li><li>回到老鼠笼子里时，他们有恢复意识充足。注意:保持acufected小鼠单独笼（每笼少于5只动物）的未acufected小鼠的笼子。 </li><li>把水瓶到位，笼返回IVC（独立通风笼）机架。 </li></ol> 4.术后护理<ol><li>有关步骤后的第一个48个小时，密切监控任何不适，包括行为变化（坐立不安，搅动或进食障碍）或外观异常（粗头发涂层或驼背姿势）小鼠。 </li></ol> IL-15ΔE7-acufected皮肤5.咪喹莫特（IMQ）处理注:转录表达和蛋白质生产acufected基因是第3天小鼠的兴趣质粒可转染后3天不同类型的兴奋剂来治疗acufected检测。我们举例说明这里通过用IMQ霜IL-15ΔE7-acufected小鼠皮肤。 IMQ是imidazoquinolin胺批准˚F或治疗外生殖器和围产期疣13。局部治疗IMQ示出以诱导人银屑病样皮肤病症与片状皮肤，表皮增殖和真皮中性粒细胞浸润12，14，15的表现小鼠。 注意:所述的质粒载体包含全长小鼠IL-15的cDNA，延伸因子1α的调节（PEF），具有在C末端（PEF-IL-的IL-2的信号肽和FLAG标签侧翼下15，5859个碱基对），将其作为由班福德等人所描述构造。 16。该质粒被用作IL-15模板删除第一16个氨基酸在残基33 - 通过SOE 48在IL-15基因，IL-15ΔE7（PEF-IL-15ΔE7，5811个碱基对）的外显子7（合成通过重叠延伸）PCR方法17。细菌菌落已成功用IL-15ΔE7转化体通过限制性酶消化验证，随后通过DNA测序12,18。在协议中步骤2所述制备无内毒素的质粒DNA的大量。 <ol><li>由三溴乙醇（每克体重400微克）的腹膜内注射麻醉acufected小鼠和眼睛应用兽医眼科软膏，以防止麻醉下干燥。检查脚趾捏反射的执行程序开始前，要确保麻醉足够的深度。注意:由于2厘米×2cm的皮肤将用于IMQ进行处理，2个剂量的pIL-15ΔE7质粒DNA（10微克每剂量10μLPBS）的被acufected 2个靶位点（1 1cm×1cm的每个）。 </li><li>标记侧翼皮肤（2厘米×2cm）上覆盖所述acufected区域。 </li><li>用棉签涂抹上标记的表面积局部涂抹奶油IMQ。注意:步骤5.1，只在第一个剂量（60毫克/剂量）进行。接下来连续的剂量适用于非麻醉小鼠。 </li><li>用高清晰度摄像机每天记录IMQ治疗背部皮肤的变化。注意:一个人持有，而另一个正在录制鼠标。静态图片拍摄，并在以后的时间编辑。 </li><li>通过注入，然后在治疗IMQ后第4天或7天颈椎脱位三溴过量的（每克体重800微克）安乐死的小鼠。皮肤切除并处理（步骤4.1 - 4.5）。 </li></ol> 6.匀浆小鼠皮肤<ol><li>使用一对外科剪刀，使从尾的基部的水平切割。双边前往后肢的基地，并继续沿侧翼前肢的底座垂直切割。 </li><li>小心剥离从后部到前部下面的组织的皮肤上。使用剪刀剪下背部皮肤。放置在无菌培养皿皮肤。 </li><li>用手术刀切除目标皮肤并在液氮立即冻结它。注意:acufected皮肤无相同的尺寸（1厘米×1cm）的或与治疗IMQ（2厘米×2cm）上是固定的，每只小鼠以最小化实验对实验变体。 </li><li>将冷冻的皮肤组织在与手柄和杵预冷的2室砂浆的中心。使用的砂浆铅锤粉碎组织。 </li><li>粉碎的组织快速放置于含有细胞裂解试剂500μLRNA提取或到含有PBS的50μL和1x蛋白酶抑制剂混合物，以获得蛋白裂解液的管子的新管中。 </li></ol> 7. H＆E和免疫组化皮肤科染色<ol><li>将皮肤组织在录像带并在4％多聚甲醛浸泡在一夜之间。 </li><li>嵌入和部分组织，这对我们来说是由病理实验室在LAC，台大进行。注意:部分被切割，其厚度为5μm。地方组织secti附件上带正电的幻灯片。 </li><li>加热载玻片在65℃下15分钟。 </li><li>请将幻灯片染色架。沉浸齿条成相应的含二甲苯替代5分钟两次，接着在100％，95％，80％，75％，60％和50％乙醇（每次5分钟），用于补液顺序浸渍槽中。浸泡在自来水中的幻灯片染色前3分钟。 </li><li>执行苏木和伊红染色。注意:在LAC，NTU在病理实验室根据请求进行H＆E染色。 </li><li>对于免疫组织化学染色，框在室温下用封闭溶液区段5分钟以减少非特异性背景染色。注:该块解决方案与商业免疫技术开发。没有动物血清被包含在该产品。 </li><li>浸在PBS中的滑动来冲块溶液。 </li><li>稀释在PBS块溶液在1:10和添加2％胎牛血清（FBS）（稀释溶液）。 </li><lI&gt;移液器的适当稀释的初级抗体到所述部分和孵育在室温下60分钟。注:抗体在稀释液稀释。分别为200 Ki67的（克隆SolA15）和Ly6G（克隆1A8），:抗体浓度为这种染色已经在1优化:800和1。这些浓度被发现不显示背景染色明显的污渍。 <ol><li>染色在同一个运行一个串行部分用相同的溶液省略初级抗体，以控制用于次级抗体的非特异性结合。 </li></ol></li><li>洗涤，每次5分钟滑动在TBS三次加0.1％吐温20（TBST溶液）。洗涤之间变化解决方案。 </li><li>移液器标记的聚合物的每个部分中的一个降和在室温下孵育40分钟。注意:标记的聚合物在商业上通过结合用过氧化物酶和第二抗体的氨基酸聚合物的量减少到Fab"片段制备。该试剂已准备好使用小鼠组织切片的免疫组织化学染色。 </li><li>洗涤滑动在TBST中三次，每次5分钟。洗涤之间变化解决方案。 </li><li>吸管3,3&#39;- diaminobenzindine四盐酸盐（DAB）溶液到组织切片，并让在室温下放置直到棕色沉淀下一个亮视野显微镜是可见的。注意:时间DAB在过氧化物酶的存在沉淀大约30分钟。 </li><li>浸泡幻灯片水停止过氧化物酶活性。浸幻灯片在核染色甲基绿的解决方案。洗涤载玻片在PBS中3次，每次5分钟。 </li><li>放置在覆盖有永久安装平台（每部分8μL）染色的截面的盖玻片。 </li></ol> 8.照片彩绘组织切片分析<ol><li>可视化亮视野显微镜下H＆E染色和IHC的组织切片。使用附连到显微镜电荷耦合器件（CCD）照相机捕获的图像。对于q分析的定量的，成像软件可用于<ol><li>扫描使用scanscope与20倍的目标整个染色切片。 </li><li>测量所述表皮的1毫米段内的表皮厚度和段内计数的Ki67免疫反应性细胞的数量。 使用显微镜自动化及图像分析软件进行定量分析:注意。表皮厚度由基底层和表皮的最外层之间的距离限定。 </li><li>计数Ly6G免疫反应细胞在皮肤区域（230 X 270微米2矩形）的正下方的局部IMQ处理皮肤的数量。 </li></ol></li></ol>

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N., DeFilippis, A. P., Azimi, N., Kurys, G., Waldmann, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5'+untranslated+region,+signal+peptide,+and+the+coding+sequence+of+the+carboxyl+terminus+of+IL-15+participate+in+its+multifaceted+translational+control.">The 5' untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control.</a> J Immunol. 160, 4418-4426 (1998).</li><li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77, 61-68 (1989).</li><li>Tan, X., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+IL-15+isoforms+generated+by+alternative+splicing+are+expressed+in+the+intestinal+epithelium.">Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.</a> Genes Immun. 7, 407-416 (2006).</li><li>Basner-Tschakarjan, E., Mirmohammadsadegh, A., Baer, A., Hengge, U. 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作者没有财务权益披露。

以确保acufected质粒DNA的表达中最关键的步骤是均匀振荡并松开皮肤的角质层。同时轻轻推针不切开皮肤，用力要够硬踏面。为了便于在上1厘米×1cm的表面积10μLDNA溶液吸收，针应当振荡在30秒为约100倍向上和向下。可以确定需要刺破皮肤上的力和通过注意的是需要得到acufected基因的最佳表达水平的次数。 修饰质粒DNA的量施加到达到表达的令人满意的程度，可能需要acufection后，因为它可以为不同的基因变化。滴定实验应当执行，以确定要在实验前以及施用质粒DNA的最佳量。从定量RT-PCR分析结果将有助于确定邻质粒DNA的量ptimal并获得可再现的结果的最佳状态。 所述acufection过程的精确机制仍有待阐明。而从局部递送的质粒DNA的转录发生在剃毛和脱掉的皮肤为低电平，通过增强转染效率（ 图3B）针灸针刺。通过microseeding协议的类比，其中与纹身枪11的多个穿孔，acufection递送的质粒DNA可能通过细胞膜角质形成细胞和毛囊19，20中的刮擦容易。通过脱毛，脱毛试剂和针摆动表皮的刺激能诱导角质形成细胞活化在一定程度上和由朗格汉斯和DC 6进一步增强的DNA摄取。此协议的一个限制是，质粒DNA的量溶于通过细胞可能不被精确地控制。我们不建议使用这种方法来治疗大面积皮肤。为了最大限度地减少在实验中所递送的基因的表达水平的变化，所述DNA溶液的体积和目标皮肤表面的大小应该被固定为相同的组实验。要生成可重复的结果，这是可取的，重复的实验是由同一个人来执行。 相对于基因枪装置和microseeding，使用针灸针束的递送裸DNA是便宜的。该程序也很简单。它需要很少的技术经验，以跟随acufection协议。 Acufection导致创伤小动物和无减肥手术后发现的。由不存在acufection操作之后显著行为变化的判断，疼痛的电平为低。 总之，使用针灸针松开由宿主细胞在皮肤DNA摄取的角质层提供了一种经济的替代方法来表达在小鼠皮肤的蛋白质。免疫相关的基因中广泛仍有待功能特征的皮肤21，这突出了对皮肤的新基因的快速学习的需要。采用这种方法，可以容易地确定感兴趣的基因的产物是否具有任何新颖功能。此acufection协议提供在调节免疫应答以治疗皮肤疾病为新发现一种方便可行工具。

皮肤是机体防御的第一线。角质形成细胞（KCS）在人类和小鼠的皮肤中的主要细胞类型。响应环境刺激（ 例如阳光，氧气，化学品和病原侵入），枯否细胞被活化并产生宽促炎细胞因子和趋化因子如IL-8，IL-6，IL-1α，IL-1β，TNF-α的阵列α和GM-CSF 1。他们一起引发免疫细胞在皮肤的招聘。加重皮肤炎症常常与许多人类疾病，包括急性异位接触性皮炎，慢性T细胞介导的炎症（ 例如过敏性接触性皮炎和银屑病）2相关联。通过抑制KC活化调节皮肤炎症反应是治疗皮肤炎症一个可行的办法。本协议描述了一种新方法，瞬时表达在表皮细胞因子基因，以研究免疫RESponse继发于皮肤这样的处理。 表皮组成的皮肤最表面的一层。它作为一个物理屏障保持外部物质如核酸和病原体进入皮肤的更深层这样。一些无针技术已经建立了表皮DNA转移3，4。在溶液中的DNA或与阳离子脂质体或腺病毒载体相关联的已被直接施加到与技术修饰表皮如除去角质层的或与脱毛试剂的治疗。角化上皮的去除促进DNA交叉的表皮屏障，并与角质形成细胞和朗格汉斯细胞相互作用以诱导免疫应答5。虽然大的表面积可用于DNA转移而这种做法不涉及针，也有缺点，包括要求对于大批量DNA（10 - 100微克），在皮肤上的苛刻处理通过汽提，并且在没有诱导DNA递送增压器6在免疫动物的免疫应答的结果不一致。裸DNA到皮肤的微粒介导的细胞内递送已被证明是高效的和可再现的用于诱导免疫应答7,8。一种手持式基因枪已被用于轰击皮肤目标部位用质粒DNA包被的金颗粒直径微米大小2通过加压氦气气流9的装置。质粒DNA被想必在皮肤采取了由KCsand树突细胞（DC）和蛋白质被局部表示或树突10被输送到引流淋巴结。尽管基因枪系统简单，需要有限的技术经验，为准备和提供的"DNA BUL成本让"（ 即金粉末，氦气）和基因枪本身限制了它的一般应用。另外，当实验在不同的位置进行了氦气输送系统的要求限制了便于基因枪交通工具。Microseeding已经证明，以实现基因转移比单次注射和粒子轰击11的更高的效率。Microseeding使用纹身枪无需使用微粒珠递送DNA至皮肤11。振荡上使用该方法对皮肤的微针是有可能直接刮细胞膜，从而在同一时间传送到DNA多个小区。然而，随着大量注射用0.254毫米直径针相关的疼痛是不利的。 在这里，我们提供一种替代的方法来在体内 DNA递送。该"acufection" </str翁&gt;我们在这里描述的方案提供了一种经济和有效的方式来在小鼠皮肤递送质粒DNA。简言之，将10针灸针（直径0.2mm×13毫米长）在通过胶粘带束被束缚。将10μL的PBS用含有感兴趣的转基因的质粒DNA的10微克体积置于剃毛，脱毛霜处理的侧面上的皮肤。通过使用针刺针束产生振荡的皮肤表面（1厘米×1cm）的，在角质层的角蛋白被松开，并且施加到所述表面上的质粒DNA被吸收进入表皮。皮肤损害进行监测，发现是最小的。在acufected皮肤转基因的表达通过定量实时PCR（定量RT-PCR）和ELISA证实。使用IMQ处理的小鼠模型12 acufection递送IL-15ΔE7对皮肤炎症的免疫调节效果进行了论证。虽然acufection证明是一个简单的，少DEMAnding方法用于体内 DNA递送，DNA为不同的基因和时代刺破皮肤的最佳量必须仔细优化以获得可再现的结果。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accu Handy Needle</td>
			<td>Accu</td>
			<td>36Gx0.5</td>
			<td>Medical device</td>
		</tr>
		<tr>
			<td>NairTM Lotion</td>
			<td>Church &#38; Dwight</td>
			<td>N/A</td>
			<td>Use to remove hair</td>
		</tr>
		<tr>
			<td>Shandon Xyline substitute</td>
			<td>Thermo Fisher Scientific</td>
			<td>6764506</td>
			<td>Deparaffinization</td>
		</tr>
		<tr>
			<td>Avertin (2,2,2-Tribromethanol)</td>
			<td>Sigma-Aldrich</td>
			<td>T4,840-2</td>
			<td>Anesthesia</td>
		</tr>
		<tr>
			<td>2-methyl-2-butanol</td>
			<td>Sigma-Aldrich</td>
			<td>152463</td>
			<td>Solvent for the dissolution of avertin</td>
		</tr>
		<tr>
			<td>DifcoTM LB broth, Miller</td>
			<td>BD</td>
			<td>244620</td>
			<td>Propagation and maintenance of E. coli for molecular biology</td>
		</tr>
		<tr>
			<td>QIAGEN&#9415; Plasmid Midi Kit</td>
			<td>QIAGEN</td>
			<td>12143</td>
			<td>Purification of plasmid DNA from E. coli</td>
		</tr>
		<tr>
			<td>Mouse IL-15/IL-15R Complex ELISA Ready-SET-Go kit</td>
			<td>eBioscience</td>
			<td>88-7215</td>
			<td>Measure IL-15 protein</td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G</td>
			<td>BioLegend</td>
			<td>127601</td>
			<td>Antibody used for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>anti-mouse Ki-67</td>
			<td>eBioscience</td>
			<td>14-5698-80</td>
			<td>Antibody used for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>Simple Stain Mouse MAXPO (Rat)</td>
			<td>Nichirei Biosciences</td>
			<td>414341F</td>
			<td>Reagent to block background signal in mouse-on-mouse stain</td>
		</tr>
		<tr>
			<td>DAB Peroxidase (HRP) Substrate Kit</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Peroxidse substrate for immunohistochemical stain</td>
		</tr>
		<tr>
			<td>Ultra V block</td>
			<td>Thermo Fisher Scientific</td>
			<td>TA-060-UB</td>
			<td>Reduce nonspecific background staining</td>
		</tr>
		<tr>
			<td>Methyl green</td>
			<td>Sigma-Aldrich</td>
			<td>M8884</td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>Vecta MountTM</td>
			<td>Vector Laboratories</td>
			<td>M-5000</td>
			<td>Permanently preserving histochemical stains</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablets</td>
			<td>Roche</td>
			<td>04-693-116-001</td>
			<td>Inhibit protease activity in cell lysate</td>
		</tr>
		<tr>
			<td>Aldara cream</td>
			<td>3M Parmaceuticals</td>
			<td>N/A</td>
			<td>5% imiquimod cream</td>
		</tr>
		<tr>
			<td>Bessman Tissue Pulverizer</td>
			<td>Spectrum Labs</td>
			<td>189475</td>
			<td>Use to pulverize skin tissue</td>
		</tr>
		<tr>
			<td>ABI7900HT cycler</td>
			<td>Thermo Fisher Scientific</td>
			<td>N/A</td>
			<td>quantitative real-time PCR assay</td>
		</tr>
		<tr>
			<td>Camcorder</td>
			<td>Panasonic</td>
			<td>HDC-SD60</td>
			<td>Image documentation</td>
		</tr>
		<tr>
			<td>Axio Scope.A1</td>
			<td>ZEISS</td>
			<td>N/A</td>
			<td>Bright-field microscopy</td>
		</tr>
		<tr>
			<td>ScanScope&#9415; XT</td>
			<td>Aperio</td>
			<td>N/A</td>
			<td>Whole-field image scanner</td>
		</tr>
		<tr>
			<td>MetaMorph&#9415; Research Imaging</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td>Quantitative analysis of skin thickness and immune-reactive cells</td>
		</tr>
	</tbody>
</table>

development of an economical dna delivery system by "acufection" and its application to skin research

在皮肤的免疫反应的失调与许多人的皮肤疾病有关。免疫相关基因直接转移到皮肤组织是一个令人着迷的方法来研究人类疾病的小鼠模型皮肤炎症的免疫调节。这里，我们提出具有成本效益的协议，它在小鼠皮肤递送裸DNA，并导致转基因表达。该方法被精压"acufection"，表示ACU穿刺介导的DNA转染 。为了执行acufection，小鼠皮肤首先用DNA注入在磷酸盐缓冲盐水（PBS），然后与针灸针束轻轻一竖以促进DNA的吸收和转染到细胞中。质粒DNA被推测采取了由角质形成细胞和皮肤中的树突细胞（DC），并表示成蛋白质。用针本身的机械刺没有造成皮肤损伤或诱导角质细胞活化。表达方式转染基因的是在皮肤在以下acufection 2天转录和翻译水平检测和维持达7天。这种acufection方法发展的主要目标是调查IL-15的先前未定义的亚型。使用这种方法，一个可变剪接IL-15同种型有部分缺失的外显子7（IL-15ΔE7）是在皮肤中表达，随后与Toll样受体7（TLR7）激动剂，咪喹莫特（IMQ）处理，以诱导炎症。 Acufection递送皮肤IL-15ΔE7抑制角质形成细胞增殖，表皮厚度和中性粒细胞募集中IMQ诱导的皮肤炎症。与在确定皮肤炎症的调节机制越来越大的兴趣，所述这里描述的协议提供了一种成本效益的和通用的替代基因枪系统或microseeding用于体内 DNA递送。这可能潜在地允许一个新颖的功能的发现基因在皮肤或调查的用于皮肤疾病的新疗法。

虽然一些发红与针灸针刺后的第一个小时之内是明显的，皮肤清除在第2天和穿刺（ 图2A）后，观察到高达至第7天无不良性皮肤反应。穿刺针与未经处理的皮肤（ 图2B）相比，H＆E分析诱导表皮正角化和最小的真皮浸润的非常低的水平。表皮厚度（ 图2C）和Ki67 +细胞（ 图2D）的数量分别为针一竖和未处理的皮肤之间是相当的，这表明针灸针一竖皮肤没有造成在表皮明显组织破碎或细胞增殖。相较于注射器针（1.067毫米直径）12，与针灸针刺趋化因子CXCL的表达诱导降低的水平-1和促炎细胞因子IL-6在第2天，持续的向第5天（ 图3A）降低。为了验证在小鼠皮肤中的acufected转基因的表达，将表达IL-15的cDNA的质粒在IL-15缺陷型C57BL / 6（IL-15 KO）小鼠的皮肤acufected。虽然在脱毛皮肤中观察到从acufected质粒DNA转录水平低，振荡与针灸针增强基因的转录水平（ 图3B）。第2天，检测IL-15的生产和保持稳定长达7天（ 图3C）。这些结果表明，acufection有效灌输的质粒DNA，并允许在皮肤中编码蛋白质的表达，而不诱导角质形成细胞过度活化。 WT小鼠用的质粒编码的IL-15ΔE7acufected。小鼠表达IL-15ΔE7mRNA和蛋白质，直到3转染后（ 图4A）天。将小鼠然后用局部IMQ软膏治疗第3天而在控制WT小鼠IMQ处理诱导银薄片，IMQ诱导的银薄片在IL-15ΔE7降低acufected WT小鼠（ 图4B）。有趣的是，acufected IL-15ΔE7的表达抑制显著+ Ly6G嗜中性粒细胞浸润到IMQ处理皮肤与unacufected WT皮肤（ 图4C）12进行比较。通过acufection，我们证明IL-15ΔE7的皮肤新颖功能。 IL-15ΔE7调制IMQ诱导中性粒细胞浸润。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55206/55206fig1.jpg" /> 图1。 针灸针的插图。 （A）的针刺针（36克×0.5&quot; ）是由一个处理和针头。 （B）十针灸针结合使用粘合带束。束的长度是如由标尺（下面板）测量大约4cm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55206/55206fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55206/55206fig2.jpg" /> 图2。 通过针灸针刺不会导致显着的表面损坏或角质细胞激活。 （A）三位点标志着鼠标的剃毛和脱掉的侧面皮肤上灰色正方形通过针灸针一竖每100次。照片是刺来监控皮肤损伤后采取的第一个小时和7天。剃毛，无针挑脱毛的皮肤被用作控制。 &lt;强&gt;（B）H＆E的来自未处理的或针状的刺小鼠皮肤福尔马林固定，石蜡包埋的皮肤切片的分析。比例尺:100μm左右。 （C）来自未处理或针一竖小鼠皮肤H＆E染色的切片的一个毫米段被选择用于测量表皮厚度（从基础到最外层的距离）。每个条代表从3个皮肤切片测得的表皮厚度的平均值。误差棒代表平均值（SEM）的标准误差。组间差异的显着性通过非配对t检验。相同的样品（D）的连续切片用抗Ki67染色用于免疫组织化学分析。每个条代表在每1mm段长度每150微米的表皮局部的Ki67 +细胞的平均。误差棒代表平均值（SEM）的标准误差。组间差异的显着性是由两个因素方差分析FOLL测试由邦费罗尼事后检验欠款。 （NS:不显著）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55206/55206fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55206/55206fig3.jpg" /> 图3。 Acufection有效表示兴趣的蛋白质，而不会引起注意的角质细胞激活。 （A）光头和脱掉的侧面皮肤保持未处理（NTC）中，用毫米直径的1.067（19 G）的注射器针头（苏尔。）研磨或通过针灸针刺的100倍（ACU）。 CXCL-1和IL-6的第2天第5天的转录水平通过测量 定量RT-PCR（N = 3）。 （B）除毛和IL-15缺陷小鼠的脱毛皮肤局部用表达IL-15的质粒DNA的液滴施加withou吨（脱毛）或针刺穿刺针（脱毛+刺）。每一组第2天在IL-15的转录物通过的TaqMan定量RT-PCR测量。 IL-15的转录是IL-15缺陷小鼠的PBS对照（NTC）不可检测的。 （C）从IL-15缺陷小鼠皮肤IL-15的生产，这是未处理的或IL-15-acufected关于各种天（每天的一个组织）通过IL-15 ELISA测量。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55206/55206fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55206/55206fig4.jpg" /> 图4。 IL-15ΔE7示范减弱IMQ诱导银片状皮肤，抑制中性粒细胞浸润。 （A）的转录水平和蛋白质生产的IL-15ΔE7-acufected（WT /的pIL-15 [6; E7）或空载体对照（WT / pEmpty）皮肤通过qRT-PCR和ELISA（n = 2的每个时间点）分别测量。 （B）在第3天空载体-或IL-15ΔE7-acufected侧翼皮肤局部用IMQ奶油（60毫克）处理。在IMQ处理后6天WT / pEmpty和WT /弼15ΔE7鼠标侧面的照片。 （C）从WT / pEmpty或WT /弼15ΔE7小鼠天3和6皮肤切片IMQ处理之后用抗Ly6G抗体染色和计数。每个条代表Ly6G +细胞从4 IMQ处理的小鼠的平均值。误差棒代表平均值（SEM）的标准误差。组间差异的显着性通过双向ANOVA然后Bonferroni 事后检验。图A和C被修改许可从杂志调查皮肤科，许可证号3901890227597.的<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55206/55206fig4large.jpg" target="_blank">请点击</a>此处查看该图的放大版本。

Watch this Scientific Journal Video about Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research at JoVE.com

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

这个协议是用于在小鼠皮肤中表达裸质粒DNA具有成本效益的选择。该协议的总体目标是提供免疫相关基因植入皮肤组织划定的皮肤炎症特定基因的功能作用。

通过"Acufection"和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

development-an-economical-dna-delivery-system-acufection-its

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.4K 次观看.  National Taiwan University College of Medicine. 这个协议是用于在小鼠皮肤中表达裸质粒DNA具有成本效益的选择。该协议的总体目标是提供免疫相关基因植入皮肤组织划定的皮肤炎症特定基因的功能作用。 

视频: Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

热酚水提取纯化及革兰氏阴性菌脂多糖的可视化

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

通过胞内细菌，研究内溶酶体系统的改造荧光纳米粒子中的应用

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

在肠系膜淋巴管空心​​大鼠模型:应用肠淋巴药物运输的评估

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

种植<em&gt; Heligmosomoides Polygyrus:</em&gt;免疫调节线虫寄生虫及其分泌产物

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

抗原驱动结肠炎模型的开发由抗原呈递细胞T细胞抗原研究的介绍

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...