小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型
1Department of Surgery,Jichi Medical University, 2Department of Surgery,University of Pittsburgh School of Medicine, 3Department of Pediatrics,University of Pittsburgh School of Medicine, 4Department of Pathology,University of Pittsburgh School of Medicine, 5Pittsburgh Liver Research Center,University of Pittsburgh, 6McGowan Institute for Regenerative Medicine,University of Pittsburgh
Summary
在这里, 我们提出了部分胆管结扎作为一个外科模型的肝损伤和再生的啮齿动物。
Abstract
在啮齿动物中, 胆总管完全胆管结扎术 (cBDL) 是研究梗阻性胆汁淤积症和胆管增生的一种成熟的外科技术。然而, 长期实验可能导致发病率和死亡率的增加。在选择有潜在肝病的小鼠菌株中, 甚至可以通过结扎肝单叶来进行有意义的比较, 这样可以减少动物的损失和开支。在这里, 我们描述的部分胆管结扎 (pBDL) 在小鼠, 其中只有左肝胆管结扎, 导致胆道梗阻的左叶, 而不是其余的裂片。通过仔细的显微外科技术, pBDL 实验可以是成本效益, 因为 unligated 叶作为一个内部控制的结扎裂片, 当受到相同的条件在同一动物。不像 cBDL, 一个单独的假操作控制组是不必要的。pBDL 是非常有用的直接比较局部和系统性的影响胆汁淤积症和其他保留胆汁成分。pBDL 也可以作为一种新的方法来研究与药物和细胞迁移有关的机制。
Introduction
外科模型的急性肝损伤和修复, 如部分肝切除或胆总管结扎 (cBDL), 已被用于研究肝再生和病理1,2。在 cBDL, 胆总管 (排泄所有肝脏产生的胆汁) 结扎, 导致梗阻性胆汁淤积, 炎症和纤维化在手术后的前2-3 周, 最终进展为肝硬化3。胆管增生, 转分化和诱导肝祖细胞也是 cBDL4,5的特征。虽然 cBDL 阐明了阻塞性 cholangiopathy 的具体机制, 其特点是在野生型小鼠中具有可重现的结果, 在这些菌株与正常肝脏6,7,8,在某些转基因小鼠肝脏疾病模型中, 由于胆道梗阻的发病率和死亡率随着时间的推移而增加, 需要的动物比预期的要多。这项技术也有利于研究具有潜在肝病的转基因小鼠, 否则可能无法容忍 cBDL 后长时间课程实验的压力。
pBDL 可以提供一个合理的替代方案来帮助克服其中的一些挑战。野生型小鼠的初步 pBDL 研究旨在区分炎症的局部作用和系统性介质9。在 pBDL 中, 胆总管上方右、左肝胆管的主胆汁汇合处仔细地可视于脐, 只结扎左肝胆管, 产生局部梗阻性胆汁淤积。pBDL 为 cBDL 提供了一些优势。在 pBDL 中, unligated 右叶作为结扎左叶的一个相同的内部控制, 受到相同的系统性影响, 如营养状况或循环因素。虽然全身性的炎症介质存在, 大泽et等) 观察到较少的纤维化和坏死的 unligated 裂片, 但增加炎症细胞和转化生长因子β表达在结扎叶 9.
最近, pBDL 被重新定义为研究保留胆汁的肝脏实质作用10。通过使用同样的动物进行内部控制 (unligated “假”) 和实验组, pBDL 有效地将每项实验所需的动物数量减半, 这反过来又更具成本效益。小鼠对 pBDL 的耐受效果好得多, 在结扎叶中可实现理想的时间过程实验 (≥2周)。最重要的是, pBDL 可以区分梗阻性胆汁淤积症和保留胆汁成分的直接肝内效应, 与 unligated 控制叶相比。总的来说, pBDL 是研究肝局部胆汁淤积症叶特异效应的一种很有吸引力的方法。
Protocol
所有程序都是按照匹兹堡大学机构动物保育和使用委员会的道德准则进行的。 1. 基本技术和一般程序 所有的显微外科设备使用前。 为了避免麻醉杀死老鼠, 仔细检查它的呼吸模式。 尽可能少接触器官和肠道, 以避免损害器官和造成出血。用湿棉签或纱布海绵按压出血点。 用湿棉签对器官进行温和的操作。湿的, 无纺布纱布海绵用于两个大小, 3.5 x 3.5 厘米和 6.0 x…
Representative Results
对于这一典型的实验, pBDL 在三 PiZ 转基因小鼠中进行, 概括了 alpha-1 胰岛素 (A1AT) 缺乏中发现的人肝病。PiZ 小鼠 overexpresses 一种转基因, 由 DNA 的多个基因组片段组成, 其中包含突变体的人类 A1AT 基因和启动子的编码区域, 以及上游和下游侧翼区域的 2 kilobases14, 15。正常 A1AT 是一种分泌的血清蛋白在肝脏中产生。A1AT 的突变体产生?…
Discussion
虽然 cBDL 是阻塞性胆汁淤积症最常见的实验技术, 但它可以在啮齿动物中提出多种挑战。根据完成终点所需的术后天数, 随着时间的推移, 动物的发病率和死亡率会显著下降。在一些转基因小鼠肝病模型的作用下, 组织损伤和胆道坏死的程度进一步恶化, 表现为肝实质异常和基线功能。与 cBDL 相比, pBDL 的报道是有限的, 但这种技术可以作为选择实验的替代品, 以减少负面结果和动物损失。
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The authors have nothing to disclose.
Acknowledgements
Khan 博士承认来自 NIH/发育研究院 K12HD052892、Alpha-1 基金会、基金会和匹兹堡肝脏研究中心的赠款支持。Michalopoulos 博士承认来自 NIH/NIDDK P01DK096990 的赠款支持。我们感谢安妮的出色和慷慨的技术援助。我们还感谢大卫. 盖勒博士提供了进入移植/啮齿动物显微外科实验室的机会。
Materials
| Microscope-Leica Wild M650, 6–40× magnification | Leica Microsystems | n/a | |
| Tec 3 isoflurane funnel fill vaporizer | General Anesthetic Services | n/a | |
| Stevens Tenotomy Scissors | Accurate Surgical & Scientific Instruments Corporation | ASSI.9193 | |
| Adson Forceps | Fine science tools | 11006-12 | |
| Mosquito classic delicate hemostatic forceps | Codman | 30-4472 | |
| Micro-retractor | Murdock; Roboz Surgical Instrument | RS-6550 | |
| Nonwoven gauze sponges | Fisherband | 870-PC-DBL | |
| Puritan 6” Small Cotton Swab w/Wooden Handle | Puritan Medical Products Company | 868-WCS | |
| Dumont SS Forceps – Standard Tips/Straight/Inox/13.5cm | Fine science tools | 11203-23 | |
| Dumont SS Forceps – Standard Tips/Angled 45°/Inox/13.5cm | Fine science tools | 11203-25 | |
| Vannas Spring Scissors – 3mm Cutting Edge | Fine science tools | 15000-00 | |
| Microneedle holder | Aesculap Surgical Instruments | FD231R | |
| Micro AROSuture, sterile 10-0 nylon suture, 70 µm, TAP points | AROSurgical Instruments Corporation | T4A10N07 | |
| 4-0 Vicryl | Ethicon | J662H | |
| 5-ml Syringe | BD | 309646 | |
| Falcon tissue culture dish, 60 × 15 mm | Corning | 353002 | |
| Isoflurane, United States Pharmacopeia (USP) liquid for inhalation, 250 ml | Piramal Healthcare | NDC 66794-017-25 | |
| Buprenex injectable (buprenorphine hydrochloride) | Reckitt Benckiser Pharmaceuticals | NDC 12496-0757-1 | |
| Cefazolin for injection , USP | APP Pharmaceuticals | NDC 63323-238-61 | |
| PVP scrub solution (povidone–iodine 7.5%) | Medline | NDC 12496-0757-1 | |
| 70% Ethanol | Decon Labs | 2716 | |
| Phosphate Buffered Saline Without Calcium or Magnesium | LONZA | 17-516F | |
| Sirius Red | Sigma | 365548 | |
| Hematolxylin | Fisher (Richard-Allan Scientific) | 22-050-111 (7211) | |
| Eosin | Anatech (Fisher) | 832 (NC9686037) | |
| Formalin | Fisher | SF100-20 |
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Citer Cet Article
Yokota, S., Ono, Y., Nakao, T., Zhang, P., Michalopoulos, G. K., Khan, Z. Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis. J. Vis. Exp. (133), e56930, doi:10.3791/56930 (2018).