使用小鼠化疗引起的粘炎模型评估小肠损伤和适应的重要端点和增殖标记
Summary
在这里,我们提出了一个协议,建立重要的终点和小肠损伤和补偿性增殖,使用化疗引起的粘炎模型。我们演示了使用细胞周期特定标记和使用小肠道重量、墓穴深度和绒痛高度作为端点来检测增殖细胞。
Abstract
肠道适应是当肠道因创伤而丧失时发生的自然补偿机制。适应性反应,如密码细胞增殖和增加营养吸收,在恢复中至关重要,但了解甚少。了解适应性反应背后的分子机制对于促进营养素或药物的识别以增强适应性至关重要。在整个文献中,都描述了不同的方法和模型,但需要一种详细的描述性方法来基本执行这些程序,以获得可重现的数据。在这里,我们描述了一种使用小鼠化疗引起的粘炎模型来估计小肠损伤和补偿性增殖的重要终点和增殖标记的方法。我们演示了使用细胞周期特定标记以及使用小肠道重量、墓穴深度和绒痛高度作为端点来检测增殖细胞。所述方法中的一些关键步骤是小肠的切除和称重,以及为测量该技术而建议的相当复杂的软件系统。这些方法的优点是它们不费时,而且具有成本效益高,易于执行和测量。
Introduction
肠道适应是自然补偿机制,当肠道因疾病或手术1,2而丧失时发生。创伤后,肠道经历形态和功能适应性反应,其特征是隐细胞增殖和增加营养吸收3。这一步骤在复苏中至关重要,但了解不足。肠道适应性反应的实验研究侧重于小鼠、大鼠和猪小肠切除后发生的变化,但了解其他损伤(如化学)自适应反应背后的分子机制或细菌)对于促进营养素或药物的识别以增强适应性至关重要。在实验中,用不同的方法来描述小肠病理学的复杂分子和细胞指数,包括组织病理学评分和损伤结果的测量。尽管如此,文献中缺少的是如何执行获取可重现数据所需的过程的详细描述。当确定适应所涉及的因素(如肠道激素)时,需要一种简单、低成本和可重复的动物模型,在这里我们建议使用化疗引起的肠道粘炎 (CIM) 模型。
损伤和适应的最简单和非常丰富的终点之一是测量小肠(SI)的质量。我们知道,粘炎的一个标志是肠细胞凋亡,时间依赖性阴萎缩和减少的丝质炎。因此,在临床前模型4、5中,检查肠道形态具有高度相关性。在人类中,血浆胆碱的下降,一个功能的肠细胞的标记,与毒性分数和炎症标记6相关,除了吸收能力7,这表明这种氨基酸是一个优秀的生物标志物粘炎。胆碱可以在小鼠和大鼠身上进行测量,并且与绒带长度8、密码生存9和辐射引起的粘炎10表现出优异的相关性。
测量等离子胆碱的一个主要优点是能够收集一种动物的重复测量。然而,小鼠的多次血液取样仅限于每周总血量为6μL/g,需要一般麻醉。不幸的是,这也限制了在小鼠中使用胆碱测量。此外,测量胆碱需要高性能液相色谱11,12,这是昂贵和费时。最近,我们发现小鼠的胆碱水平与SI重量(p<0.001)(未公布的数据)显著相关,使胆碱成为反映肠细胞质量的直接测量。测量SI重量的一个限制是必须牺牲小鼠,因此在同一小鼠内不可能重复测量。尽管如此,该方法提供了对研究问题进行各种其他组织分析的可能性,这些事实可以想象弥补对动物的额外使用。因此,我们建议使用SI体重作为小鼠损伤和适应的一种简单、低成本和快速的生物标志物。为了确保可重复性和可接受的分析变异,应小心地从动物中取出肠道,用盐水冲洗,在称重前排空和干燥。在本文中,我们将准确介绍如何执行此过程。
粘炎的另一个特征是墓穴中增殖细胞的丧失,以及再生期3期间补偿性超增增。细胞标记Ki67经常被用来通过免疫组织化学13来确定快速增殖细胞。尽管Ki67是扩散的简单标志,但它有不精确的倾向,因为Ki67在细胞周期的所有活跃阶段(G1、S、G2和M)14中都存在。特定标签对于检测复制细胞至关重要,这就是为什么我们建议原位加入5-bromo-2′-脱氧尿氨酸(BrdU),一种合成的胸腺素模拟物,因为它在很大程度上仅限于在S-阶段15中复制细胞。BrdU在牺牲前150分钟注射到动物体内,随后可以使用BrdU特异性抗体通过免疫组织化学检测细胞。在该方法文章中,我们展示了如何使用自由图像软件测量墓穴中BrdU免疫阳性细胞的面积。
形态和功能变化通常在5-FU诱导粘炎模型中进行研究,其中肠道适应是通过绒痛高度和墓穴深度进行评估的。在这项研究中,我们发现,在粘炎的急性阶段(等于损伤阶段)中,BrdU合并所测量的增殖与墓穴深度无关。与此相反,在诱导后3至5天,墓穴深度与粘炎修复阶段出现增殖显著相关。这表明粘炎的急性阶段不能单靠墓穴深度来测量。我们建议,当在粘炎小鼠的急性阶段使用增殖作为终点时,最好使用BrdU合并,但是当在再生阶段的后期定量增殖时,密码深度是合理的替代BrdU公司。本研究的目的是描述这个模型的方式,它可以被所有研究人员使用,无论是在肿瘤学领域,特别是研究人员不熟悉肠道损伤模型。
所述模型可用于根据自适应响应,使用体重、SI重量和墓穴深度作为终点进行表型转基因模型。例如,我们在这里展示我们如何在L细胞分泌不足的细胞敲除模型中使用5-氟尿酸(5-FU)诱导粘炎的模型。胰高血糖素样肽-1(GLP-1)和胰高血糖素样肽-2(GLP-2)是肠道激素,从肠内分泌L细胞中共同分泌,以回应食物摄入量17,18。GLP-2被认为是肠道愈合、粘膜凋亡调节和改善SI 19、20、21、22阻隔功能的重要因素。根据文献资料,我们假设内源性激素对于损伤后适应性反应中发生的补偿性超增至关重要。
Protocol
Representative Results
Discussion
在这里,我们演示了一种在小鼠模型中研究SI损伤和再生的可广泛访问方法。存在多种肠道损伤的临床前动物模型,但我们必须了解,每种模型都是独一无二的,并且端点必须适合回答研究问题。该模型非常适合研究对损伤的自适应反应,但在将该模型用作粘炎临床前模型时,应修改端点。然而,从动物模型到病人的翻译是具有挑战性的23。我们建议的SI重量和扩散的终点应仅限于自适应响…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了诺和诺德基础代谢研究中心和伦德贝克基金会的无限制资助。
Materials
| 5-Fluorouracil | Hospira Nordic AB, Sweden | 137853 | |
| Ketaminol®Vet | Merck, New Jersey, USA | 511485 | |
| Rompun®Vet Xylazine | Rompunvet, Bayer, Leverkusen, Germany. | 148999 | |
| 10% nautral formalin buffer | Cell Path Ltd, Powys, United Kingdom | BAF-5000-08A | |
| HistoClear | National Diagnostics, United Kingdom | HS-200 | |
| Pertex | HistoLab®, Sweden | 840 | |
| BrdU | Sigma-Aldrich, Germany. | B5002 | |
| Tris/EDTA pH 9 buffer | Thermofisher scientific, Denmark | TA-125-PM4X | |
| Peroxide Block | Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark | TL-060-QHDM | |
| Rodent Block buffer | Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark | TL-060-QHDM | |
| Monoclonal mouse anti-BrdU antibody | Thermofisher Scientific, Denmark. | MA1-81890 | |
| Lab Vision Antibody Diluent OP Quanto | Thermofisher Scientific, Denmark. | TA-125-ADQ | |
| Horseradish peroxidase | Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark | TL-060-QHDM | |
| DAB Quanto Substrate | DAB Substrate Kit, Thermofisher Scientific, Denmark | TA-125-QHDX | |
| DAB Quanto Chromogen | DAB Substrate Kit, Thermofisher Scientific, Denmark | TA-125-QHDX | |
| Zen Lite Software (Blue edition) | Carl Zeiss A/S | https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html | |
| ImageJ Software | LOCI, University of Wisconsin | https://imagej.nih.gov/ij/ |
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