JHN 得到瑞士国家基金会 (SNSF 310030_146290) 的支持。

在巴塞尔大学生物医学系 (瑞士巴塞尔) 的动物设施中, 小鼠被饲养并保存在特定的无病原体 (SPF) 条件下。所有的老鼠实验都是按照瑞士联邦和州的规定进行的 (动物协议编号 2816 [巴塞尔莫斯丹塔])。
1. 脂多糖溶液的制备
<ol><li>打开从大肠杆菌中纯化的 LPS 0111: B4 在无菌条件下, 并在水中重新将其重组为5毫克/毫升的浓度. </li>
	<li>稀释含无菌磷酸盐缓冲盐水 (PBS) 的 LPS 库存, 使其工作浓度为0.2 µg/µL。</li>
</ol>2. 腹腔注射 LPS 对小鼠的作用
<ol><li>在层流气流中用30克针吸入0.5 毫升注射器中的 LPS 工作溶液。</li>
	<li>称雌性 C57Bl/6 小鼠 (六岁至10周), 并计算将注射的 LPS 量:10 µL (2 µg)/g 体重。 注: 例如, 如果鼠标重20克, 则20克 x 10 µL = 200 µL LPS 工作溶液需要注射。</li>
	<li>轻轻而坚定地处理鼠标, 一方面抑制动物。确保动物能够正常呼吸, 但不能在克制的时候扭动和转动。</li>
	<li>将鼠标的鼻子稍稍倾斜到地板上, 露出腹部注射。定位腹部中线和从中线左或右低位腹部注射部位。向控制小鼠注入 ip PBS 而不是 LPS。</li>
	<li>用自由的手, 注入适当的体积 (在步骤2.2 中计算的体积) 的 LPS 工作解决方案。确保针进入腹膜腔, 否则, 可能会出现腹腔肌肉层的错误注射。不过, 不要太深的针进入腹腔, 以避免伤害内器官位于该地区。</li>
	<li>把动物小心地送到笼子里, 每笼多达5只老鼠。</li>
</ol>3. 监控小鼠
<ol><li>在注射时对动物进行监测, 每2小时注射8小时, 以检测内毒素血症的发生和严重程度。因此, 使用已从以前发布的手稿24改编的评分表 (表 1)。</li>
	<li>
分数的 LPS 注射小鼠的以下参数表1所列
	<ol>
<li>检查通常光滑的毛皮的外观。如果毛皮出现褶皱, 尖刺或肿胀的皮毛, 表明不适和/或疼痛, 评分为 1, 2, 或3。</li>
		<li>检查鼠标的活动。 注意: 老鼠通常在整个笼子周围自由走动进食, 饮酒, 攀爬, 但抑制或限制活动可能是痛苦的征兆。</li>
		<li>检查意识水平。 注意: 老鼠很好奇, 他们通常在笼子里四处走动调查周围。缺乏或减少运动会提示疼痛和不适。</li>
		<li>检查眼睛 注: 完全睁开眼睛有望在健康的小鼠, 但部分或完全闭合的眼睛, 可能与分泌物, 可以表明疼痛。</li>
		<li>检查呼吸速率。 注意: 健康的老鼠通常有正常和快速的呼吸速率, 降低呼吸率可能是不适的征兆)。</li>
		<li>检查呼吸质量。 注意: 呼吸困难或不喘气是痛苦的征兆。</li>
	</ol>
</li>
</ol>4. 核酸 (RNA) 提取和均匀化的组织取样
<ol><li>准备收集/均匀化管: 添加1毫升单步 RNA 隔离试剂到2毫升管预先填入1.4 毫米陶瓷球。</li>
	<li>在适当的时间点 (前 (0 小时) 和2小时, 4 小时和8小时后, LPS 注射液) 弄死小鼠与麻醉过量 (异氟醚) 后放血和进行解剖。</li>
	<li>用剪刀切开皮肤和肌肉层, 用内脏把腹腔露出来。</li>
	<li>仔细解剖脾脏、肝脏和结肠, 将器官从脂肪中清除, 并将它们放在 PBS 上放在冰上。</li>
	<li>用剪刀或手术刀从每个器官剪下一小块 (大约0.5 厘米长)。 注意: 在每只老鼠 (肝脏的同一裂片、近端或远端结肠) 中, 总是从器官的同一部位取一块, 这一点很重要。</li>
	<li>将纸巾放在纸上, 然后将其放在收集/均匀化管中, 确保它完全浸入 RNA 隔离试剂中。</li>
	<li>融汇高速台式均质体中的组织。对于像脾脏、肝脏和结肠这样的软组织, 在三十年代的速度6.00 中使用1的均匀化循环。</li>
	<li>在室温下孵化样品5分钟。</li>
	<li>小心地将管子放在液氮中, 将组织的裂解液凝固。将快照冷冻裂解液贮存在-80 摄氏度, 直到 RNA 分离。</li>
</ol>5. 从组织中提取 RNA
注意: RNA 分离试剂、氯仿和醇被认为是危险物质。在化学罩下进行 RNA 提取。使用经认证的无 RNase 试剂和设备, 以保持 RNase 的环境。
<ol><li>
相分离
	<ol>
<li>在冰上解冻冰冻组织裂解液。</li>
		<li>离心样品在 1000 x g 5 分钟, 在4°c, 以颗粒的剩余的组织微粒, 可能会留下。</li>
		<li>将上清液转移到新的无核酸酶1.5 毫升管。</li>
		<li>每1毫升 RNA 分离试剂中加入200µL 的冰冷氯仿, 用手用力摇动10-15 秒。</li>
		<li>室温下孵育2-3 分钟。</li>
		<li>离心机在 12, 000 x g 15 分钟在4°c。 注: 样品现在包含3个阶段: 低红苯酚-氯仿相, 界面和上无色水相。RNA 在上部水相存在。</li>
		<li>收集上水相 (总容积的 50%), 并转移到一个新的无核酸酶1.5 毫升管。</li>
	</ol>
</li>
	<li>
RNA 降雨雪
	<ol>
<li>每1毫升 RNA 分离试剂加入0.5 毫升冰冷异丙醇, 并通过反转管5-6 次轻轻混合。</li>
		<li>室温下孵育10-15 分钟。</li>
		<li>离心机在 1.2万 x g 10 分钟在4摄氏度。 注意: RNA 颗粒在这个阶段应该是可见的。</li>
	</ol>
</li>
	<li>
RNA 颗粒的洗涤
	<ol>
<li>完全去除含有异丙醇的上清液, 不干扰颗粒。</li>
		<li>用1毫升冰冷75% 乙醇清洗颗粒, 每1毫升 RNA 分离试剂用于初始裂解。</li>
		<li>简单地涡流样品。</li>
		<li>离心样品在 7500 (或 1.2万) x g 5 分钟在4°c。</li>
	</ol>
</li>
	<li>
溶解 RNA
	<ol>
<li>完全去除上清液中的乙醇而不干扰颗粒。</li>
		<li>将开管置于化学罩下3-5 分钟, 使 rna 颗粒在室温下风干 (不要过度干燥, 因为在这种情况下很难溶解 rna)。</li>
		<li>在20-50 µL 核酸酶水中溶解 RNA 颗粒, 吹打小心上下。</li>
		<li>在55摄氏度孵化样品10-15 分钟, 进一步改善 RNA 的溶液。 注意: 在这个阶段, RNA 可以储存在-80 摄氏度, 直到进一步分析。</li>
	</ol>
</li>
</ol>6. 剩余 DNA 的消化
<ol><li>通过将整除 (2 µl) 吹打到分光光度计中, 测量 RNA 浓度, 并调整到推荐浓度。</li>
	<li>开始消化与最大的污染 DNA。10µg RNA 在50µL 核酸酶无水。</li>
	<li>添加0.1 卷 10x DNase i 缓冲器和1µL rDNase 我每样, 并轻轻地混合吹打上下。</li>
	<li>孵育37°c 20-30 分钟。</li>
	<li>涡流 DNase 灭活剂, 并添加0.1 体积的样品与吸管混合良好。</li>
	<li>有时用手在室温下混合孵育5分钟。</li>
	<li>离心机在 1万 x g 1.5 分钟。</li>
	<li>将含有无 DNA RNA 的上清液转移到新的无核酸酶管中。</li>
	<li>用分光光度法测定 RNA 浓度和质量。</li>
</ol>7. 反向转录
<ol><li>使用商业上可用的脱氧核糖核酸 cDNA 反向转录 (RT) 试剂盒进行反向转录。 注: 在这里, 多达2µg 的 RNA 可以逆转转录。</li>
	<li>计算体积, 其中包含2µg RNA。吸管2µg RNA 在一个新的 PCR 管。</li>
	<li>将核酸酶的水添加到 PCR 管中的样品中, 最终体积为10µL。</li>
	<li>通过混合表 2中列出的下列组件, 准备2x 主混合
</li>
	<li>添加10µL 2x 主混合到10µL RNA 获得1x 主混合在总容量20µL。</li>
	<li>关闭聚合酶链反应 (PCR) 管, 并简单地向下旋转样品。</li>
	<li>将示例放在 PCR Thermocycler 中, 并设置表 3中列出的设置。</li>
	<li>将生成的 cDNA 存储在-20 摄氏度以进行进一步分析。</li>
</ol>8. 定量 PCR (qPCR)
<ol><li>使用商业上可用的工具包执行 qPCR 反应。</li>
	<li>自设计和测试内含子生成底漆, 使用前, 具有不同浓度的模板 cDNA。通过创建标准曲线来分析底漆的功效, 并使用高效、正确的熔融曲线的引物。 注意: 本实验中使用的引物序列列在表 2中。</li>
	<li>用表 4中描述的反应设置, 准备384井板中的定量聚合酶链反应 (qPCR) 反应。</li>
	<li>用铝箔把所有的溶液和盘子放在冰上, 以防在反应混合物准备好的时候使盘子变湿。</li>
	<li>执行 triplicates 中的所有反应。</li>
	<li>使用表 5中列出的设置在 thermocycler 上执行 PCR 反应。</li>
	<li>计算 triplicates 所有反应的平均 Ct 值。通过计算 2 ^ (-ΔCt) 将所有目标基因正常化为 glycerinealdehyde-3-phosphate-dehydrogenase (Gapdh)家政基因的表达水平。 注意: 所有的目标基因, 平均 Ct > 35 被认为是在检测限额。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+colitic+C3H/HeJBir+mice+demonstrate+selective+antibody+reactivity+to+antigens+of+the+enteric+bacterial+flora.">Spontaneously colitic C3H/HeJBir mice demonstrate selective antibody reactivity to antigens of the enteric bacterial flora.</a> J Immunol. 159, 44-52 (1997).</li><li>Macpherson, A. J., McCoy, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardised+animal+models+of+host+microbial+mutualism.">Standardised animal models of host microbial mutualism.</a> Mucosal Immunol. 8, 476-486 (2015).</li><li>Masopust, D., Sivula, C. P., Jameson, S. 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,+Dirty+Mice,+and+Men:+Using+Mice+To+Understand+Human+Immunology.">Of Mice, Dirty Mice, and Men: Using Mice To Understand Human Immunology.</a> J Immunol. 199, 383-388 (2017).</li><li>Wirtz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+lethal+septic+peritonitis+by+neutralizing+the+biological+function+of+interleukin+27.">Protection from lethal septic peritonitis by neutralizing the biological function of interleukin 27.</a> J Exp Med. 203, 1875-1881 (2006).</li></ol>

作者没有什么可透露的。

该协议模仿微生物衍生产品入侵后发生的免疫学过程。该协议中的关键步骤是选择鼠标线、小鼠的卫生状况、脂多糖的剂量、监测动物对内毒素血症的发生情况以及实验终止的时间点。最重要的是, 必须考虑到小鼠菌株的遗传背景。不同的小鼠菌株对 LPS 有不同的敏感性。例如, C3H/HeJ 和 C57BL/10ScCr 小鼠对 LPS 诱导的内毒素血症有抵抗力21,25。此外, 动物的卫生状况可能影响 LPS 内毒素血症的病程。特异的无病原体 (SPF) 动物是最常用的。这些老鼠是从一份已定义的病原体中解脱出来的, 但是这些动物的完整的微生物组成是未知的26,27。无菌的无菌或无菌动物 (不窝藏微生物群) 有别于 SPF 鼠 26.可能, 无菌动物与一个微生物或一个微生物联合体 (悉生动物) 的殖民化可能会影响基因的表达, 如 IL-19, 在 LPS 注射液与 SPF 动物相比,8。此外, 有一个评分表被认为是用来作为一个评分表的脓毒症模型诱导的粪液注入腹腔腔24。这 sheetcan 将与当地动物福利委员会讨论, 并可适应当地的需要。特别是, 需要与当地动物福利委员会讨论终止的标准。这项实验必须停止, 当分数 > 12 达到或当一个单独的参数的最大得分达到。在这个实验中, 八只动物中没有一个动物在注射 LPS 后超过12的疾病评分 (2 µg/g 体重)。在本研究中, 结果显示细胞因子Il6, Il1b, TNFa和Il10在肝, 脾和结肠注射 LPS 后的表达。Il1b、 TNFa和Il10在脾、肝和结肠内注入 lps 后达到4小时的表达, 而Il6表达在脾、结肠和4小时后肝内毒素注射后达到2小时。在 8 h 中, 表达式Il6、 Il1b、 TNFa和Il10返回到脾脏、肝脏和结肠的基线水平。当Il6、 Il1b、 TNFa和Il10的表达式已返回到基线级别, 表明Il6的表达增加时, 在 LPS 注射液后6小时和10小时之间的疾病严重程度达到峰值,Il1b、 TNFa和Il10在 lps 注射液后迅速发生, 但其他基因的动力学可能在 lps 注射液后显示出不同的模式。对这些细胞因子的表达也可以通过 ELISA 的方法对动物血液中的蛋白质水平进行分析, 除 qPCR 外还可以考虑。
在达到疾病评分 > 12 的情况下, 需要对该技术进行修改和故障排除。因此, 应减少脂多糖的剂量, 并更好地调整以使用的鼠标线和当地条件, 以避免过早终止实验。通过删除特定的基因组区域或 overexpressing 基因来操纵基因, 可能会影响小鼠对 LPS 的敏感性。在初步实验中, 脂多糖的剂量可以滴定适当的剂量, 以避免对动物的不必要的伤害和死亡的实验。值得注意的是, 必须避免对 LPS 与其他细菌衍生产品的污染和错误注射。此外, 需要确定脂多糖注射液后感兴趣基因表达的动力学。
限制是, 这项技术提供了关于在肠道屏障破坏后将微生物产品传播到宿主的后果的信息, 但不会透露导致肠道屏障破坏的事件的信息, 并导致那会引发肠病当然, 这个模型不是一个结肠炎模型作为 DSS 结肠炎模型, 但它可以是有用的除了结肠炎模型, 以研究的后果, 微生物产品进入宿主。此外, 内毒素的 ip 注射将扰乱腹腔, 其中小和大肠位于。这可能会促进肠道源细菌产品从腹膜腔转移到宿主。
这个模型的意义在于它使用了一个定义的玉兰。在其他模型中, 包括全细菌的 ip 注入、粪便解决方案的 ip 注入24或脓毒性腹膜炎模型, 其中腹膜炎是由盲肠结扎和穿刺28引起的, 大量的微生物或其产品诱发疾病。此外, 在小鼠体内注射 LPS 是一种简单的方法, 不需要手术, 如盲肠结扎和穿刺引起的脓毒性腹膜炎。
该模型的进一步应用是研究, 目的是调查内毒素血症或 LPS 引起的败血症, 在任何情况下的特点是由细菌衍生产品转运到宿主, 如违反皮肤屏障或违反了泌尿生殖道。总之, 这是一个简单的模型, 可用于每个实验室设置。根据当地情况, 可能需要对当地需求进行适应。特别是, 在进行实验之前, 必须与地方当局讨论评分表。当细菌或细菌衍生产品在发生屏障破坏后进入宿主时, 这种方法被用来模拟宿主的后果。这可能是特别相关的研究, 检查的基础上, 肠道屏障破坏是一个常见的事件在疾病的病理。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>
One of the authors&#39; names was corrected from:
Katarina Raduolovic
to:
Katarina Radulovic

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/57610/injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial">Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach</a>.&#160;An author name was updated.

Erratum: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

人类肠道被殖民化, 形成了微生物群, 在进化过程中与宿主建立了互惠互利的关系。在这种关系中, 宿主为微生物群提供了一个安全的利基, 而微生物群则提供维生素, 养分消化和保护病原体到宿主, 其中微生物群驻留在1。当宿主和微生物群之间的这种有益关系受到干扰时, 疾病就会发生, 如炎症性肠病 (IBD)。IBD 是由遗传和环境因素引起的肠道慢性炎症性疾病, 发生于两种主要形式, 克罗恩病 (CD) 和溃疡性结肠炎 (UC)。尽管两种 IBD 形式有相似之处, 但它们的特点是炎症修饰的位置和性质存在一定的差异。CD 是一种复发性壁炎症紊乱, 可能扩展到胃肠道的任何部分, 而 UC 是非壁的, 并限于结肠。此外, 核苷酸结合齐聚域含蛋白 2 (NOD2) 的突变, 一种模式识别受体 (PRR), 识别 muramyl 二肽 (MDP), 一个组成部分的细胞壁的大多数革兰氏阳性和阴性细菌, 是与 CD2关联。此外, 大肠杆菌(大肠杆菌)、李斯特菌和链球菌及其产品都在 CD 患者的巨噬细胞中发现, 在屏障破裂3后进入宿主。当细菌或其产品在 CD 的研制过程中进入宿主时, 免疫系统就会产生一种反应, 从而导致生产循环抗菌抗体4。也许, 最有说服力的证据表明, 微生物在 IBD 发病机制中的作用源于老鼠模型。当动物被抗生素治疗, 或当老鼠被保存在无菌 (GF) 条件下, 疾病的严重性在大多数结肠炎模型中减少, 如在 GF 设施中不开发结肠炎的 IL-10-/小鼠5,6。此外, 结肠炎也扰乱了微生物群的组成, 其特点是不平衡的组成和减少的丰富称为 dysbiosis7。IBD 的结果可能是增加肠道通透性, 从而导致微生物和微生物源产品进入宿主。
在动物中, 葡聚糖硫酸钠 (DSS) 的应用诱导肠道上皮破裂, 导致上皮屏障的通透性增强8。门内毒素的浓度在动物中升高, 与 DSS 结肠炎9。有趣的是, 缺乏 C 型凝集素受体特异细胞内黏附分子 molecule-3 抓取 nonintegrin 同源相关 1 (SIGN-R1) 的动物保护不受 DSS 结肠炎和 LPS 诱导的内毒素血症10。为了进一步传播到宿主, 细菌或细菌衍生产品必须通过血管屏障11, 腹膜腔, 在其中的小和大肠, 肠系膜淋巴结和/或肝脏12。为了降低系统的复杂性, 采用了一种定义的细菌衍生化合物。lps, 导致内毒素血症后腹腔 (ip) 或静脉注射 (静脉注射) 注射13注射到小鼠, 研究 interleukins Il6和Ilb和细胞因子Tnfa响应 LPS 的表达。
lps 是一种病原体相关分子模式 (玉兰), 表达为革兰阴性菌的细胞壁成分, 由脂 a (lps 结构中的主要玉兰)、核心寡糖和 O 侧链14组成。由树突状细胞、巨噬素和上皮细胞表达的4类像样受体 (TLR4) 识别 LPS15, 这需要联合受体进行适当的结合。急性期蛋白脂多糖结合蛋白 (LBP) 束缚脂多糖形成一个复杂的转移脂多糖到分化 14 (CD14), 一个 glycosylphosphatidylinositol 定位膜蛋白。CD14 进一步航天飞机 LPS 到淋巴细胞抗原96或也称为 MD-2, 这是与细胞外领域的 TLR4。LPS 与 MD-2 的结合促进了 TLR4/MD-2 的二聚化诱导构象变化, 以吸收细胞内的适配器分子激活下游信号通路14, 其中包括髓系分化初级反应基因 88 (MyD88) 依赖通路和包含适配器诱导干扰素β (TRIF) 依赖通路16。TLR4 对 LPS 的识别然后激活 NF-nf-κb 通路, 诱导炎症细胞因子的表达, 如 TNFα、IL-6 和 IL-1β17。
特别是当 lps 被注射到动物体内时, 应考虑到动物的脂多糖浓度、动物的遗传背景和饮食。高浓度的脂多糖导致感染性休克, 其特点是低血压和多脏器衰竭, 最后到死亡18。与人类相比, 小鼠对 lps 的敏感性较小, 其中脂多糖浓度介于 2-4 ng/千克体重之间 (生物武器) 能够诱发细胞因子风暴19。对于小鼠, 致命剂量 (LD50), 导致死亡的一半的老鼠范围从10-25 毫克/千克体重20取决于使用的鼠标应变。对于常用的小鼠菌株, C57Bl/6 和 BALB/c, 致死剂量 50% (LD50) 为10毫克/千克。相比之下, C3H/HeJ 和 C57BL/10ScCr 的菌株受到 LPS 诱导的内毒素血症的保护, 这是由于Tlr421中的突变造成的。因此, Tlr4-deficient 小鼠 hyporesponsive 注射 LPS22。其他基因修饰的鼠标线, 如 PARP1-/鼠23对 LPS 诱导的毒性休克有抵抗力。
这里描述的老鼠模型使用致死剂量的 lps 进行系统的管理, 以模拟在屏障破坏身体表面后, lps 传播的后果。所选择的 LPS 浓度 (2 毫克/千克) 没有诱发 C56Bl/6 小鼠的死亡率, 而是诱导释放的促炎性细胞因子。

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DreamTaq Green PCR Master Mix (2x)</td>
			<td style="width:303px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:119px;">K1081</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High Capacity cDNA Reverse Transcription Kit,</td>
			<td style="width:303px;">Applied Biosystems, Foster City, CA, USA</td>
			<td>4368813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-Free DNase Set,</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS Escherichia coli O111:B4</td>
			<td>Invivogen, San Diego, CA, USA.</td>
			<td>tlrl-eblps</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnican 50 Single-use insulin syringe</td>
			<td>B. Braun Melsungen, Melsungen, Germany</td>
			<td>9151125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bioanalyzer 2100</td>
			<td>Agilent Technologie, Santa Clara, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge 5430</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge Mikro 220R</td>
			<td>Hettich, Kirchlengern, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection tools</td>
			<td>Aesculap, Tuttlingen, Germany</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast-Prep-24 5G Sample Preparation System</td>
			<td>M.P. Biomedicals, Santa Ana, CA, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop ND-1000</td>
			<td>NanoDrop Products, Wilmington, DE, USA</td>
			<td style="width:119px;">not applicable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRI Reagent</td>
			<td>Zymo Research, Irvine, CA, USA</td>
			<td>R2050-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

injections of lipopolysaccharide into mice to mimic entrance of microbial-derived products after intestinal barrier breach

肠道上皮屏障将宿主与通常耐受或忽略的微生物群分离开来。这种屏障的破坏导致细菌或细菌来源的产品进入宿主, 进入宿主循环和内脏, 导致炎症性肠病 (IBD) 患者观察到无控制发炎, 即的特点是肠道上皮通透性增加。
为了模拟细菌源性化合物进入宿主体内的入口, 采用了内毒素血症模型, 并将脂多糖 (LPS) 作为革兰氏阴性菌外细胞壁的一部分注入小鼠。在本研究中, 致死剂量的 LPS 被腹腔注射, 并随后监测的小鼠8小时使用疾病评分。此外, 在不同时间点的 LPS 注射后, 在脾、肝、结肠中分别分析了炎症细胞因子Il6、 Il1b、和Tnfa的表达水平。该模型可用于研究在侵入微生物或细菌衍生产品后对机体表面的屏障破坏引起的免疫应答。

为了模拟在肠道屏障破坏后发生的细菌或细菌衍生产品进入后宿主的后果, LPS 在致死剂量 (2 µg/克体重) 中注入 C57Bl/6 小鼠体内。每只老鼠都被监测并得分, 其中包括: 小鼠的出现, 动物的活动, 眼睛的状况, 和呼吸速率和质量 (表 1), 其中含有内毒素血症的发生和评分表中列出的参数。.这些动物显示了疾病的临床症状, 在注射 LPS 后6至10小时达到峰值, 在24小时内恢复 (图 1)。单个小鼠在注射 LPS 后牺牲了2小时、4小时和8小时。从脾脏、肝脏和结肠中采集组织切片。RNA 被分离了从这些器官和反向转录到 cDNA。该 cDNA 用作 qPCR 的模板, 用于确定Il6 (图 2A) Il1b (图 2B)、 TNFa (图 2C) 和Il10 (图 2D) 的表达式级别, 以及用于 qPCR 的引物。在表 6中列出。在注射脾脏和结肠后, Il6的表达达到峰值 2h, 肝脏注射后4小时。Il1b、 TNFa、和Il10的表达式在注入脾脏、肝脏和结肠后达到4小时的峰值。在注入后8小时内, Il6、 Il1b、 TNFa、和Il10的表达式返回到基线级别。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57610/57610fig1.jpg"> 图 1: LPS 注射液 (2 µg/克体重) 诱发 C57Bl/6mice 内毒素血症的临床征象.注射2µg 体重的 LPS 后, 个别小鼠被监测的疾病评分表 1, 其中包括动物的外观和活动, 他们的眼睛睁开, 他们的呼吸率和质量每2小时12小时和 24 h 船尾er 脂多糖注射液。疾病评分 (y 轴) 被涂抹到各自的时间 (x 轴)。给出了4-5 只小鼠每一个时间点的平均 * SD。<a href="/files/ftp_upload/57610/57610fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57610/57610fig2.jpg"> 图 2: LPS 注射液 (2 µg 体重) 诱导 C57Bl/6 小鼠促炎性细胞因子的表达.注射小鼠2µg 体重的 LPS 和Il6 (A)的表达水平, Il1b (B), Tnfa (C)和Il10 (D)是由 qPCR 在脾脏, 肝脏和结肠在指示的时间分析穴位注射后。细胞因子表达水平全部规范化为Gapdh的表达式。平均每个时间点为4-5 只老鼠显示。<a href="/files/ftp_upload/57610/57610fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
表 1: 在 LPS 注射后监测 C57Bl/6 小鼠的疾病评分表.在 LPS 注入后监测的参数。在注射后每2小时对动物进行监测12小时, 并对表中列出的每一项参数给予评分。每个动物的最后得分代表了所有个体得分的总和。这与引用24相适应。<a href="/files/ftp_upload/57610/table1_LPSscore.xlsx">请单击此处下载此文件.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>量</td>
			<td>试剂</td>
		</tr>
<tr>
<td>2µL/样品</td>
			<td>10x RT 缓冲器</td>
		</tr>
<tr>
<td>0.8 µL/样品</td>
			<td>25x 核苷酸 (dNTP) 混合 (100 毫米)</td>
		</tr>
<tr>
<td>1µL/样品</td>
			<td>RT 随机引物</td>
		</tr>
<tr>
<td>4.2 µL/样品</td>
			<td>核酸酶水</td>
		</tr>
</tbody></table>表 2: 用于准备反向转录的主混合剂的试剂。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
		</tr>
<tr>
<td>25°c</td>
			<td>10分钟</td>
		</tr>
<tr>
<td>37°c</td>
			<td>120分钟</td>
		</tr>
<tr>
<td>37°c</td>
			<td>5分钟</td>
		</tr>
<tr>
<td>4°c</td>
			<td>举行</td>
		</tr>
</tbody></table>表 3: 用于反向转录的 Thermocycler 设置。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>量</td>
			<td>试剂</td>
		</tr>
<tr>
<td>5µL/井</td>
			<td>2x 主组合</td>
		</tr>
<tr>
<td>0.05 µL/井</td>
			<td>参考染料</td>
		</tr>
<tr>
<td>500毫微米决赛/井</td>
			<td>前进底漆</td>
		</tr>
<tr>
<td>500毫微米决赛/井</td>
			<td>反向底漆</td>
		</tr>
<tr>
<td>在10和100之间, 我们用了40的/好的</td>
			<td>模板 cdna 稀释在模板稀释缓冲 (1/100 稀释这个缓冲是在无核酸酶的水, 并用于稀释模板 cdna)。稀释此缓冲区中的模板可使模板添加到的井可视化, 因为反应颜色由蓝色变为绿色</td>
		</tr>
<tr>
<td>核酸酶水至10µL/井的最终容积</td>
			<td>核酸酶水</td>
		</tr>
</tbody></table>表 4: PCR 反应的描述。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td></td>
		</tr>
<tr>
<td>95°c</td>
			<td>2分钟</td>
			<td></td>
		</tr>
<tr>
<td>95°c</td>
			<td>5 s</td>
			<td rowspan="2">40个周期</td>
		</tr>
<tr>
<td>60°c</td>
			<td>二十年代</td>
		</tr>
<tr>
<td>95°c</td>
			<td>十五年代</td>
			<td rowspan="3">熔融曲线分析</td>
		</tr>
<tr>
<td>60°c</td>
			<td>1分钟</td>
		</tr>
<tr>
<td>95°c</td>
			<td>十五年代</td>
		</tr>
</tbody></table>表 5: 用于 PCR 的 Thermocycler 设置。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆</td>
			<td>序列</td>
			<td>熔化温度</td>
			<td>产品长度</td>
		</tr>
<tr>
<td>Il6-F</td>
			<td>TCG GCT TAA TTA ATG</td>
			<td>60。3</td>
			<td>94 bp</td>
		</tr>
<tr>
<td>Il6-R</td>
			<td>GCATCATCGTTGTTCATACAATCA</td>
			<td>58。2</td>
			<td></td>
		</tr>
<tr>
<td>Il1b-F</td>
			<td>TGTGAAATGCCACCTTTTGA</td>
			<td>56。1</td>
			<td>94 bp</td>
		</tr>
<tr>
<td>Il1b-R</td>
			<td>GTCAAAGGTTTGGAAGCAG</td>
			<td>57。2</td>
			<td></td>
		</tr>
<tr>
<td>Il10-F</td>
			<td>ATCGATTTCTCCCCTGTGAA</td>
			<td>56。2</td>
			<td>108 bp</td>
		</tr>
<tr>
<td>Il10-R</td>
			<td>TGTCAAATTCATTCATGGCCT</td>
			<td>56。1</td>
			<td></td>
		</tr>
<tr>
<td>Tnfa-F</td>
			<td>CCACCACGCTCTTCTGTCTAC</td>
			<td>60。4</td>
			<td>103 bp</td>
		</tr>
<tr>
<td>Tnfa 河</td>
			<td>AGGGTCTGGGCCATAGAACT</td>
			<td>60</td>
			<td></td>
		</tr>
<tr>
<td>Gapdh-F</td>
			<td>CATCAAGAAGGTGGTGAAGC</td>
			<td>56。7</td>
			<td>199 bp</td>
		</tr>
<tr>
<td>Gapdh 河</td>
			<td>CCTGTTGCTGTAGCCGTATT</td>
			<td>58</td>
			<td></td>
		</tr>
</tbody></table>表 6: qPCR 反应中使用的底漆.用于 qPCR 检测小鼠细胞因子和管家基因的引物序列Gapdh.

Watch this Scientific Journal Video about Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach at JoVE.com

向小鼠注射脂多糖以模拟肠道屏障破坏后微生物衍生产物的进入免疫学和感染|视频

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

本文提出了一种模拟肠道屏障破坏后细菌衍生化合物入口的协议。将低致死剂量的脂多糖系统地注入小鼠体内, 对24小时注射后进行监测。在脾、肝、结肠几个时间点上测定了促炎性细胞因子的表达。

注射脂多糖对小鼠肠道屏障破坏后微生物源产物的模拟进入

The intestinal epithelial barrier separates the host from the microbiota that is normally tolerated or ignored. The breach of this barrier results in the ...

injections-lipopolysaccharide-into-mice-to-mimic-entrance-microbial

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19.3K 次观看.  University of Basel. 本文提出了一种模拟肠道屏障破坏后细菌衍生化合物入口的协议。将低致死剂量的脂多糖系统地注入小鼠体内, 对24小时注射后进行监测。在脾、肝、结肠几个时间点上测定了促炎性细胞因子的表达。

视频: Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

采用雾化脂多糖一种产生肺中性粒细胞增多

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

科研

免疫与感染

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.

母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I:C）

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Immunofluorescence Analysis of Stress Granule Formation After Bacterial Challenge of Mammalian Cells

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of infected cells, including organelle ultrastructure, cytoskeletal network organization, as well as cellular processes such as Stress Granule (SG) formation. The characterization of how pathogens subvert host processes is an important and integral part of the field of pathogenesis. While variable phenotypes may be readily visible, the precise analysis of the qualitative and quantitative differences in the cellular structures induced by pathogen challenge is essential for defining statistically significant differences between experimental and control samples. SG formation is an evolutionarily conserved stress response that leads to antiviral responses and has long been investigated using viral infections1. SG formation also affects signaling cascades and may have other still unknown consequences2. The characterization of this stress response to pathogens other than viruses, such as bacterial pathogens, is currently an emerging area of research3. For now, quantitative and qualitative analysis of SG formation is not yet routinely used, even in the viral systems. Here we describe a simple method for inducing and characterizing SG formation in uninfected cells and in cells infected with a cytosolic bacterial pathogen, which affects the formation of SGs in response to various exogenous stresses. Analysis of SG formation and composition is achieved by using a number of different SG markers and the spot detector plug-in of ICY, an open source image analysis tool.

We describe a method for the qualitative and quantitative analysis of stress granule formation in mammalian cells after the cells are challenged with bacteria and a number of different stresses. This protocol can be applied to investigate the cellular stress granule response in a wide range of host-bacterial interactions.

哺乳动物细胞细菌挑战后应激颗粒形成的免疫荧光分析

Fluorescent imaging of cellular components is an effective tool to investigate host-pathogen interactions. Pathogens can affect many different features of ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

针对天然产物的单克隆抗体的产生

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

活小鼠肠道移植物对宿主病的诱导及微内窥镜评价

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

对TNBS介导肠纤维化发展的力学洞察与拉帕霉素抑制作用的评价

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

使用人类脐带血干细胞（CB-SC）治疗后，单核细胞分化成表状独特的巨噬细胞-衍生的外体

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...