建立小鼠重症急性胰腺炎模型，将牛磺胆酸钠逆行注射到胆胰管中
1Department of Gastroenterology,the First Affiliated Hospital of Soochow University, 2MOE Engineering Center of Hematological Disease,Soochow University, 3Cyrus Tang Hematology Center,Soochow University, 4National Clinical Research Center for Hematologic Diseases,the First Affiliated Hospital of Soochow University, 5MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology,The First Affiliated Hospital of Soochow University, 6Collaborative Innovation Center of Hematology,Soochow University
Summary
本文描述了严重急性胰腺炎的小鼠模型。这里介绍的程序非常快速，简单且易于访问，因此有可能以方便的方式研究急性胰腺炎的分子机制和不同的治疗干预措施。
Abstract
急性胰腺炎 （AP），尤其是重度急性胰腺炎 （SAP） 的患病率在较年轻年龄组中呈每年增加趋势。然而，在目前的临床实践中缺乏有效的治疗方法。由于转基因和敲除菌株易于获得，并且其体积小，允许 体内 评估所需的最小剂量的药物，因此在小鼠中建立良好的实验模型是AP研究的首选。此外，通过牛磺胆酸钠（TC）诱导的SAP是目前使用最广泛和表征最好的模型之一。该模型已被研究用于AP过程中的新疗法和可能的分子事件。在这里，我们介绍了使用牛磺胆酸钠和简单的自制微注射器生成AP小鼠模型。此外，我们还为随后的组织学和血清学检测提供方法。
Introduction
急性胰腺炎（AP）是胰腺的急性炎症，其特征在于主胰管阻塞，随后导管扩张和胰腺自身消化不良。其临床表现包括局部或全身炎症、腹痛和血清淀粉酶升高1，2。根据严重程度分类3，AP可以轻度，中度和重度形式出现，其中，重症急性胰腺炎（SAP）是最令人担忧的疾病，因为它的死亡率超过30%4。在美国，AP 是住院的最常见原因之一，影响了超过 200，000 名患者5。此外，AP，尤其是SAP，每年都在增加，并影响到较年轻的年龄组6。然而，在目前的临床实践中缺乏有效的治疗方案6，7。因此，有必要探索AP中涉及的分子机制，从而促进治疗的改善。
需要完善的实验动物模型来研究AP所涉及的机制并评估不同治疗方式的有效性。由于转基因和敲除菌株易于获得，并且其体积小，从而最大限度地减少 了体内 评估所需的药物剂量，小鼠是AP研究的首选。因此，已经在小鼠中开发了几种AP模型8，9。
Niederau等人从通过静脉内给药caerulein10诱导的轻度胰腺炎大鼠模型开始，开发了一种SAP小鼠模型，该模型使用相同的药物和注射途径11诱导腺泡细胞坏死。尽管该模型具有几个优点，包括无创性，快速诱导性，广泛的可重复性和适用性，但主要缺点是在大多数情况下仅开发轻度形式的AP，从而限制了其临床相关性。酒精被认为是AP的主要病因之一;然而，Foitzik等人报告说，只有当与其他因素（例如外分泌过度刺激）结合使用时，它才会引起胰腺损伤12。此外，尽管通过不同的给药途径开发的酒精诱导的AP模型，并且已经报道了药物剂量13，14，15，但它们的主要缺点是难以重现它们。腹膜内给予l-精氨酸也可以诱导小鼠AP16;然而，其低临床相关性阻碍了其应用。牛磺胆酸盐是一种胆汁盐，由 Creutzfeld 等人于 1965 年首次提出，用于通过胰管输注诱导类似于人 AP 的疾病17。尽管在病理生理学中的临床相关性存在争议18，19，但牛磺胆酸盐诱发的胰腺炎仍然是 SAP 不可或缺的模型。
由于该模型易于实现且对小鼠也有效，因此它现在是小动物 体内 研究最常用的AP模型之一。Perides等人使用牛磺胆酸钠（TC）在小鼠中诱导SAP20，为了解其病理学提供了见解。结合基因修饰技术，该模型使我们能够确认AP中涉及的几个特定基因。例如，Bicozo等人表明，CD38基因的敲除可以防止TC输注胰腺炎模型，并将这些机制归因于细胞内Ca2 + 信号传导的改变21。Fanczal等人研究了TRPM2在小鼠胰腺泡和导管细胞质膜中的生理意义，并证明了TC诱导的SAP在TRPM2敲除小鼠中的严重程度降低22。此外，该模型还为 在体内测试许多新药提供了一种简单有效的方法。例如，该方法能够验证咖啡因23，脱氢胆酸24以及各种抗氧化剂和抗凝剂的治疗效果25，26。这一证据证明了TC诱导的SAP模型的多功能性。尽管Wittel等人描述了类似的小鼠模型27，但缺乏有关实现过程的详细信息可能导致无法重现这些发现。在本文中，我们重点介绍使用简单的自制微量注射器的方法，并研究TC诱导的SAP，从而不仅为进一步研究AP的发病机制和治疗提供了可能的指导，而且还为许多其他物质的完美适应性实验方法提供了可能的指导。
Protocol
所有涉及动物的实验均获得苏州大学动物伦理委员会的批准。所有外科手术均在完全麻醉下进行。根据以前的文献，没有使用镇痛药来避免干扰疾病的自然过程28，29。对缺乏镇痛药的认可也得到了苏州大学动物伦理委员会的批准。 1. 准备工作 在手术前8-12小时快速使用C57BL / 6野生型小鼠。 准备5%（w / v）…
Representative Results
通过仔细遵循上述说明，我们获得了大约40分钟的平均手术持续时间。小鼠略微无活性，在术后24小时，48小时和72小时分别损失了约0.5-1.75g，0.85-1.85g和0.5-4.73g的重量（图2）。 从手术完成到手术后24小时，随着疾病的发展，小鼠变得不活跃，并表现出缓慢的反应和动作。 在术后72 h，对照组和SAP小鼠的存活率分别为100%（8/8）和72.7%（8/…
Discussion
TC诱导的SAP模型是一种出色的研究工具。如本研究所示，该模型在一般实验室中非常容易实现，而无需使用特定设备。当与组织学和生化分析结合使用时，它提供了一种成本（廉价的试剂）和省时（24小时时间窗口）的方法来诱导和评估AP。调整TC的浓度也提供了产生轻度和中度AP的可能性。Perides等人还使用TC在小鼠中诱导SAP20，主要区别在于他们使用泵进行TC输注， 这可能提供更?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢以下资助的支持：NCRCH的转化研究补助金[2020WSA01]，苏州市卫生委员会青年学者KJXW科学补助金[KJXW2020002]，苏州市科学技术计划（SKY2021038和SKJY2021050），江苏省高等教育机构优先学术项目发展（PAPD）的资助，以及江苏省小学研究与社会发展计划（BE2018659）。
Materials
| 0.5% iodophor | Shanghai Likang Disinfectant | 310102 | 4 mL/mouse |
| 0.9% sodium chloride | Sinopharm Group Co., Ltd. | 10019318 | 0.8 mL/mouse |
| 1% Pentobarbital sodium | Sigma | P3761 | 0.2 -0.25 mL/mouse |
| 25 μL flat tip Microliter syringe | Gaoge, Shanghai | A124019 | |
| 4% Paraformaldehyde | Beyotime, Nantong, China | P0099-500ml | |
| 5% sodium taurocholate (TC) | Aladdin | S100834-5g | 10 μL/SAP mouse |
| 6-0 Sterile nylon microsuture with threaded needle (1/2 circle) | Cheng-He | 20093 | |
| 75% alcohol | Sinopharm Group Co., Ltd. | 10009218 | 4 mL/mouse |
| 8-0 Sterile nylon microsuture with threaded needle (3/8 circle) | Cheng-He | 19064 | |
| ALT Activity Assay Kit | EPNK, Anhui, China | ALT0012 | |
| Amylase Assay Kit | EPNK, Anhui, China | AMY0012 | |
| Angled small bulldog clamp with 12 mm jaw (3 cm) | Cheng-He | HC-X022 | |
| aspen shavings or shreds for mouse bedding | Beijing Vital River Laboratory Animal Technology | VR03015 | |
| AST Activity Assay Kit | EPNK, Anhui, China | AST0012 | |
| Blood Urea Nitrogen (BUN) Assay Kit | EPNK, Anhui, China | BUN0011 | |
| C57BL/6 mouse | Beijing Vital River Laboratory Animal Technology | 213 | |
| Creatine Assay Kit | EPNK, Anhui, China | CRE0012 | |
| Feature microtome blade | Beyotime, Nantong, China | E0994 | |
| Hemostatic Forceps (9.5 cm, Curved) | JZ, Shanghai Medical Instruments Co. Ltd. | JC3901 | |
| Lipase Assay Kit | Jiancheng, Nanjing, China | A054-2-1 | |
| Microtome | Leica biosystem, Germany | RM2245 | |
| Mindray biochemistry analyzer | Mindray, Shenzhen, China | BS-420 | |
| MPO Assay Kit | Jiancheng, Nanjing, China | A044-1-1 | |
| Normal mouse chow | Trophic, Nantong, China | LAD 1000 | |
| Phosphate buffered saline | Beyotime, Nantong, China | C0221A | |
| Straight micro-bulldog clamp with 5 mm jaw (1.5 cm) | JZ, Shanghai Medical Instruments Co. Ltd. | W40130 | |
| Straight or curved forceps (11.0 cm) | Cheng-He | HC-X091A or HC-X090A | |
| Straight Scissors (10.0 cm) | Cheng-He, Ningbo, China | HC-J039102 | |
| Thermo Scientific Centrifuge | Thermo Scientific, USA | Multifuge X1R |
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Citar este artigo
Zhou, X., Chen, H., Wei, X., He, Y., Xu, C., Weng, Z. Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct. J. Vis. Exp. (182), e63129, doi:10.3791/63129 (2022).