牛磺胆酸钠诱导C57BL / 6小鼠严重急性胰腺炎
Summary
严重急性胰腺炎的动物模型能够在初始阶段研究病理生理变化，促进观察炎症事件的演变。在这里，我们提供了通过将牛磺草酸钠逆行输注到麻醉的C57BL / 6小鼠的胰管中来诱导严重急性胆汁性胰腺炎的方案。
Abstract
通过牛磺胆酸钠输注诱导胆道急性胰腺炎已被科学界广泛使用，因为代表了人类临床状况和与临床胆道胰腺炎发作相对应的炎症事件的再现。胰腺损伤的严重程度可以通过测量输注胆汁酸的浓度、速度和体积来评估。本研究提供了方案生殖中使用的材料和方法的更新清单，并显示了该急性胰腺炎（AP）模型的主要结果。以前的大多数出版物都仅限于在大鼠中复制这种模型。我们已经将这种方法应用于小鼠，这提供了可能与研究相关的其他优势（即，这些动物的试剂和抗体库的可用性以及使用转基因小鼠品系的可能性）。对于小鼠急性胰腺炎诱导，我们提出了一个系统方案，在C57BL / 6小鼠中以10μL / min的输注速度以2.5%牛磺胆酸钠的定义剂量3分钟，在诱导后12小时内达到其最大严重程度，并突出结果并具有验证该方法的结果。通过实践和技术，从麻醉诱导到输注完成的总估计时间为每只动物25分钟。
Introduction
在人类中，胆结石的存在是胰腺炎的最常见原因，这是由于胆总管末端部分的阻塞，中断了胰腺分泌物的流动并导致胰腺中强烈的炎症过程，血清和炎症介质中消化酶的浓度增加1，2。
已经提出了两种不同的理论来解释急性胰腺炎（AP）的发展。”共通道”理论表明，胆囊中存在的结石会阻塞远端胆总管系统，从而使胆汁分泌逆行流入胰管。第二种理论（”导管阻塞”理论）表明，过量胆结石阻塞胰管会导致胰腺分泌物流向十二指肠的阻塞，从而导致导管高血压3。虽然导致急性胆道胰腺炎的机制尚不完全清楚，但结果是强烈的炎症过程。消化酶出疹和胰腺自身消化导致组织病理学改变，腹水和血清中炎症细胞因子（IL-1β，IL-6，TNF-α）增加，以及急性期蛋白增加4，5，6。
严重的急性胰腺炎是一种值得临床关注的疾病，因为它涉及多个器官和高死亡风险。用于急性胰腺炎（AP）繁殖的动物模型很重要，因为它们解释了疾病的病理生理机制，并有助于监测炎症事件的演变，从疾病的初始阶段开始。这在诊所中通常是不可能的2，7。此外，在临床前研究中，胰腺组织的获取很容易，有利于阐明与临床条件相关的变化8 以及使用等基因物种的可能性，消除不良变量，并反映临床与人类状况中观察到的结果的相似性9。
用于诱导大鼠和小鼠急性胰腺炎的胆道和非胆道模型已在科学文献中频繁研究。非胆道诱导方法包括给予超近端刺激剂量的胆囊收缩素促分泌剂或其类似物cerulein10;给予几乎致命剂量的l-精氨酸;或给予补充乙硫氨酸的胆碱缺乏饮食11。虽然这些方法很容易复制并导致胰腺炎症，但它们不会复制理论上触发AP的机制（即胆汁分泌物反流进入胰管）。解决胆道模型的技术是基于胆汁酸逆行输注到胰管中，需要训练有素的研究人员来执行该协议。已经发表了几项在大鼠中使用这种方法的研究（显然是出于技术原因，因为这些实验涉及外科手术）12，13。然而，在小鼠中的方法可能会在炎症研究中提供更有趣的结果3，14，15。在这项研究中，我们将展示通过输注牛磺胆酸钠在C57BL / 6麻醉小鼠中繁殖严重急性胰腺炎的步骤清单。
对于涉及需要抗体实验以及基因和蛋白质表达分析的工作，使用小鼠是优选的，因为这些动物的材料库更多，并且有可能使用等基因和敲除物种，以及其他可用于研究16。小鼠C57BL / 6是一种小鼠近交系，最初用于研究抗肿瘤活性和免疫学。该菌株越来越被研究人员视为同种异基因，允许结果具有更大的可重复性，这可能意味着在实验中使用较少数量的动物，并且同一组之间结果的变异性较小17，18。
Perides等人（2010）14 发表了一种通过牛磺胆酸钠输注在小鼠中诱导AP的方案。在这里，我们使用C57BL / 6小鼠中更高的牛磺胆酸钠浓度（2.5%）更新该模型，并具有定义的输注体积和速度（图1）。在小鼠诱导后12小时内达到最大严重程度。血清和腹膜腔中IL-6浓度的升高与AP的进展相关。通过实践，从麻醉诱导到输注完成的总估计时间为每只动物25分钟。训练有素的研究人员进行这项实验至关重要。为确保将溶液正确注射到胆总管中，请使用亚甲蓝而不是牛磺胆酸钠进行几次飞行员培训课程。
Protocol
Representative Results
Discussion
通过逆行牛磺胆酸钠输注诱导急性胰腺炎的方法已经在大鼠中显示22，23，24。分别于2008年、2010年和2015年出版的三部类似作品作为议定书的参考3，14，15。在这项工作中，我们列出了在C57BL / 6小鼠中重现该方法的所有关键步骤以及验证它的一些可能性。
<…Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢圣保罗大学医学诊所的毕业后计划;Coordenação de Aperfeiçoamento de Pessoal de Nível Superior（CAPES）和圣保罗大学医学院（FMUSP）。
Materials
| 0.4 mm needle | INTRAG MEDICAL TECH | 90183210 | 30G |
| 0.54 mm polyethylene tube | Tygon | 730010 | – |
| Styrofoam block | – | – | – |
| masking tape for mounting the mouse | Missner | 1236 | – |
| Infusion pump scheduled to 10µL / min. | Havard aparatus-Peristaltic Pump Series | MA1 55-7766 | Model 66 Small Peristaltic |
| Scissors and forceps | |||
| Antiseptic providine iodine | Pfizer | 12086OR | antisepsis |
| 70% ethanol | SIGMA | 459836 | Mix 700 mL 100% ethanol with 300 mL dH2O |
| Razor blade | Lord | bdk9a1ghk6 | For trichotomy |
| Sodium taurocholate | Sigma-Aldrich | 86339- 1G | CAS NUMBER- 345909-26-4 |
| microvessel clip | Medicon Surgical | 56.87.35 | Approximator, opening 4.0 mm, closing pressure 30 - 40 g |
| 6-0 prolene | Bioline | 5162 | Suture line |
| Ketamin NP (cloridrato de dextrocetamina) 50mg/mL | Cristália | ||
| Xilazine 2% | Syntec | ||
| Sterile saline solution (0.9% (wt/vol) saline) | Farmace | 105851 | |
| Methyl Blue | Sigma-Aldrich Chemicals | M5528 | |
| MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel – Immunology Multiplex Assay | MERCK | MCYTOMAG-70K | Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples. |
| Amylase Assay | Labtest | 11 | |
| Desmarres retractor 13-mm width |
ROBOZ | RS-6672 |
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