通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型
1The First Affiliated Hospital of Anhui University of Science & Technology, 2Medical College of Anhui University of Science & Technology, 3Endocrinology Department,The First Affiliated Hospital of Anhui University of Science & Technology, 4Department of Hepatobiliary Surgery,The First Affiliated Hospital of Anhui University of Science & Technology
Summary
腹腔注射药物给药是一种安全有效的非侵入性胰腺损伤方法。本研究比较了小鼠五种不同的腹腔注射方案诱导不同程度的胰腺损伤，并建立了严重胰腺损伤模型，以研究严重急性胰腺炎 （SAP） 的病理变化和治疗策略。
Abstract
严重急性胰腺炎 （SAP） 的治疗死亡率高，带来了重大的临床挑战。使用动物模型研究与 SAP 相关的病理变化有助于确定潜在的治疗靶点和探索新的治疗方法。既往研究主要通过逆行胆管注射他韦罗胆酸钠诱导胰腺损伤，但手术损伤对动物模型质量的影响仍不清楚。在这项研究中，我们采用不同频率的腹腔注射 Caerulein 联合不同剂量的 LPS 诱导 C57BL/6J 小鼠胰腺损伤，并比较了五种腹腔注射方案的损伤程度。关于诱导小鼠急性胰腺炎，提出了一种腹膜内注射方案，导致 5 天内死亡率高达 80%。具体来说，小鼠每天腹膜内注射 10 次 Caerulein （50 μg/kg），然后在最后一次 Caerulein 给药后 1 小时注射 LPS （15 mg/kg）。通过调整注射药物的频率和剂量，可以有效地控制胰腺损伤的严重程度。该模型表现出较强的可控性，并且复制周期短，因此可以由单个研究人员完成，而无需昂贵的设备。它方便、准确地模拟了在人类 SAP 中观察到的关键疾病特征，同时表现出高度的可重复性。
Introduction
重症急性胰腺炎的特点是消化系统疾病领域1 内起病迅速、进展迅速、死亡率高。其高病死率一直是临床研究的突出重点。由于临床状况的不可预测变化、疾病表现的异质性以及人类标本的有限可用性，建立动物模型对于疾病研究变得越来越重要。
逆行将牛磺胆酸钠注射到胆总管中通常用于创建 SAP2 的大鼠模型。通过模拟胰胆梗阻并诱导胆汁和胰液反流，这种建模技术在复制 SAP 动物模型方面表现出很高的成功率。但是，应该注意的是，侵入性手术确实对动物模型本身有影响。此外，这种方法仅限于大型动物，例如大鼠和狗，它们主要用作实验对象。替代技术，包括十二指肠插管3 （duodenal intubation）、直接十二指肠穿刺4 （direct duodenal puncture） 和胆管-胰管直接穿刺5 （direct puncture of the bile duct-pancreatic duct），经常用于建模目的。
腹膜内注射和饮食建模方法具有非侵入性优势，可应用于任何大小的动物。通过喂养胆碱缺乏乙硫氨酸 （CDE）6 诱导的 SAP 小鼠模型存在某些并发症，例如难以控制的高血糖和低钙血症，使其不适合评估新的诊断和治疗方法。另一方面，腹膜内注射 Caerulein 联合 L-精氨酸7 是诱导小鼠急性胰腺炎最常用的方法。具体来说，Caerulein（一种胆囊收缩素类似物）的重复腹膜内给药为研究与这种破坏性疾病相关的各个方面提供了一种非常合适的方法，包括发病机制、炎症和再生过程。由于其结构与胆囊收缩素 （CCK） 相似，Caerulein 可有效刺激胆囊收缩和胰酶分泌，导致酶分泌失衡，随后发生自毁8。脂多糖 （LPS） 作为一种病原体相关分子模式分子普遍存在并被广泛研究，可通过腹腔注射与 Caerulein 结合，建立有效的 SAP 小鼠模型。这种组合会迅速触发并释放大量炎性细胞因子，导致过度的局部和全身炎症。几项研究报道了通过腹膜内注射 Caerulein 联合 LPS 在小鼠中诱导 SAP 模型。这可能是由于腹腔注射 Caerulein 可引起小鼠胰腺水肿和出血，而加入 LPS 可立即诱导胰腺坏死并加剧全身炎症反应、败血症甚至器官衰竭。目前，腹膜内 Caerulein 注射的剂量和频率存在差异，额外 LPS 剂量也存在不一致。在小鼠 SAP 模型中实现一致性具有挑战性 9,10,11,12;因此，有必要建立一个标准化的协议来获得理想的模型。在本文中，我们描述了一种小鼠腹腔注射方案，并研究了 LPS 的最佳注射频率和额外剂量。
Protocol
该协议由安徽科技大学第一附属医院（中国淮南）伦理委员会审查和批准（伦理守则：2023-KY-905-001）。该研究遵循美国国立卫生研究院 （National Institutes of Health） 关于在所有动物手术中照顾和使用研究啮齿动物的指南。本研究使用体重 20-30 g 的 C57BL/6J 成年小鼠。将小鼠在受控条件下（约 21 °C，昼夜交替循环 12 小时）在动物实验室中饲养一周。小鼠在整个过程中都可以随意获取食物和水。材料 <s…
Representative Results
实验小鼠建模的过程如图 1 所示。注射完成 12 小时后，使用旷场录像机监测不同实验组小鼠的运动距离和不动持续时间 5 个周期（图 2A）。在 5 个周期中，PI V 组小鼠在 3 分钟内保持低水平的运动距离，而 3 分钟内的不动率随着随后的每个周期而增加（图 2B、C）。此外，对不同实验组小鼠在 5 个周期内?…
Discussion
目前，缺乏有效的方法来改善严重急性胰腺炎患者的高死亡率。研究药物在增强免疫稳定机制方面的疗效至关重要。迫切需要一种理想的严重急性胰腺炎动物模型。具有 C57BL/6J 遗传背景的小鼠广泛用于生物医学研究，包括 SAP 病理生理学研究。B6J 小鼠 70 多年的遗传分化导致几个外显子自发缺失15，导致对 Caerulein 诱导的胰腺损伤的敏感性降低16。此外，现有的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了淮南市健康与医学科学研究项目 （No.HNWJ2023005）;淮南市市指导科技计划项目（2023151号）;安徽省大学生创新创业训练计划（第S202310361254号）;“50·“科技之星”创新团队 淮南市和安徽省临床重点专科建设项目。感谢安徽科技大学第一附属医院检验科提供相关检测数据。
Materials
| 20× Citric Acid Antigen Repair Solution (pH 6.0) | Wuhan servicebio Technology Co.,Ltd, China | G1202-250 ml | |
| Amylase | Mindray,China | ||
| Annexin V-FITC/PI | Wuhan servicebio Technology Co.,Ltd, China | G1511 | diluted at 1:20 |
| Anti-HMGB1 Rabbit pAB | Wuhan servicebio Technology Co.,Ltd, China | GB11103 | diluted at 1:1800 |
| BCA protein quantitative detection kit | Wuhan servicebio Technology Co.,Ltd, China | G2026-200T | |
| BD FACSCanto II Flow Cytometer | BD Life Sciences, San Jose, CA, 95131, USA | BD FACSCanto II | |
| BSA | Wuhan servicebio Technology Co.,Ltd, China | GC305010-100g | |
| C57BL/6J | Cavion Experimental Animal Co., Changzhou, China | license number SCXY (Su) 2011–0003 | |
| Ceruletide | MCE, New Jersey, USA | 17650-98-5 | 50 µg/kg |
| Chemiluminescence imager | Cytiva CO.,LTD.;USA | ||
| Citric acid antigen repair Solution (Dry powder pH 6.0) | Wuhan servicebio Technology Co.,Ltd, China | G1201-5 L | |
| Collagenase IV | Wuhan servicebio Technology Co.,Ltd, China | GC305014 | 0.5 mg/mL |
| DAB (SA-HRP) Tunel Cell Apoptosis Detection Kit | Wuhan servicebio Technology Co.,Ltd, China | G1507-100 T | |
| Dimension EXL with LM Integrated Chemistry System | Siemens Healthcare Diagnostics Inc.Brookfield,USA | YZB/USA 8311-2014 | |
| ECL developer | Wuhan servicebio Technology Co.,Ltd, China | ||
| Eosin dye (alcohol soluble) | Wuhan servicebio Technology Co.,Ltd, China | G1001-100 ml | |
| EthoVision XT | Noldus, Netherlands | ||
| FITC-labeled goat anti-rabbit IgG | Wuhan servicebio Technology Co.,Ltd, China | GB22303 | diluted at 1:50 |
| Fully automatic blood cell analyzer | Zybio Inc. China | Zybio-Z3 CRP | |
| GapDH | Wuhan servicebio Technology Co.,Ltd, China | GB11103 | diluted at 1:1500 |
| Hematoxylin blue return solution | Wuhan servicebio Technology Co.,Ltd, China | G1040-500 ml | |
| Hematoxylin differentiation solution | Wuhan servicebio Technology Co.,Ltd, China | G1039-500 ml | |
| Hematoxylin dye | Wuhan servicebio Technology Co.,Ltd, China | G1004-100 ml | |
| HMGB-1 ELISA kits | njjcbio Co., Ltd, China | ||
| HOMOGENIZER | Wuhan servicebio Technology Co.,Ltd, China | KZ-III-F;IC111150 100222 | |
| HRP-labeled goat anti-rabbit IgG | Wuhan servicebio Technology Co.,Ltd, China | GB23303 | diluted at 1:1500 |
| IL-6 ELISA kits | Wuhan servicebio Technology Co.,Ltd, China | GEM0001 | |
| Lipase | Mindray,China | ||
| Lipopolysaccharide | Wuhan servicebio Technology Co.,Ltd, China | GC205009 | 15 mg/kg |
| Low temperature high speed centrifuge | Changsha Pingfan Apparatus&Instrument Co.,Ltd.,China | TGL-20M | |
| Membrane breaking liquid | Wuhan servicebio Technology Co.,Ltd, China | G1204 | |
| microtome | Jinhua Craftek Instrument Co., Ltd.;China | CR-601ST | |
| Nylon mesh | Wuhan servicebio Technology Co.,Ltd, China | 200-mesh | |
| One-step TUNEL cell apoptosis detection kit (DAB staining method) | Wuhan servicebio Technology Co.,Ltd, China | G1507-100T | |
| Paraffin tissue embedding machine | PRECISION MEDICAL INSTRUMENTS CO.,LTD;Changzhou,China | PBM-A | |
| Pathological tissue drying apparatus | PRECISION MEDICAL INSTRUMENTS CO.,LTD;Changzhou,China | PHY-III | |
| Phosphate-buffered saline | Wuhan servicebio Technology Co.,Ltd, China | G4202-100ML | |
| PMSF | Wuhan servicebio Technology Co.,Ltd, China | G2008-1 ml | |
| Positive fluorescence microscope | Olympus Corporation,Tokyo, Japan | BX53 | |
| Pro Calcitonin | Mindray,China | ||
| PVDF membrane | Millipore, USA | 0.22 µm | |
| RIPA | Wuhan servicebio Technology Co.,Ltd, China | G2002-100 ml | |
| SDS-PAGE | Beyotime Biotechnology,China | P0012A | |
| TNF-αELISA kits | Wuhan servicebio Technology Co.,Ltd, China | GEM0004 | |
| Ultrasonic water bath | DONGGUAN KQAO ULTRASONIC EQUIPMENT CO.,LTD.;China | KQ-200KDE | |
| Western Blot | Bio-Rad Laboratories, Inc.,USA | ||
| Western blot imaging System | Global Life Sciences IP Holdco LLC, JAPAN | Amersham ImageQuant 800 | |
| Whirlpool mixer | SCILOGEX;USA |
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Xu, L., Xu, M., Xie, Y., You, W., Wang, J., Xu, L., Feng, Q., Sun, J., Zhang, J., Yang, H., Qi, W. Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection. J. Vis. Exp. (207), e66780, doi:10.3791/66780 (2024).