该二甲基亚硝胺诱发肝纤维化模型大鼠
Summary
We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.
Abstract
四至六周龄，雄性Wistar大鼠用于产生肝纤维化动物模型。该方法需要四个星期10毫克/公斤的二甲基亚硝胺（DMN）施用，每周连续三天腹膜内给予。作为DMN是一个已知hepatoxin和致癌物质腹膜内注射是在通风柜进行。该模型具有若干优点。首先，肝变化可依次或在感兴趣的特定阶段的研究。其次，肝脏疾病的阶段可通过血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）的酶测定进行监测。第三，在不同阶段的肝损伤的严重程度可以通过在指定的时间点处死动物来确认，随后Masson的腺毛染色的肝组织的组织学检查。 4周DMN给药后，典型的纤维化评分是对伊沙克比例5至6。该模型可以一致地被再现，并已广泛用于评估潜在的抗纤维化剂的功效。
Introduction
DMN是一种强效的肝毒素特异性。报道了马吉1其代谢，组织分布，并造成伤害到大鼠的肝脏能力，和由Pritchard的和巴特勒2中记载的细胞凋亡的肝细胞损伤和细胞死亡的机制。该化合物的间歇给药报道以诱导在狗和大鼠3,4-肝纤维化。
该机制与肝纤维化的形态学变化，采用这种模式被广泛研究。在使用大鼠早期研究中，DMN 3周的治疗产生小叶中央出血性坏死后肝硬化结节无脂肪变性5。它表明，在早期纤维化，所形成的胶原是更多的交叉与III型挂钩比I型6更为突出。在与其他原因常见，DMN诱发纤维性变化用的增加Kupffer细胞相关联;驻留在我的肝巨噬细胞n个血窦。这些细胞演变成肌成纤维细胞，并产生细胞外基质这是纤维化7的首要问题过量。
在细胞信号方面，Nakamura等人证明了TGF-β在肝纤维化8的进展中起关键作用。他们用表达截短的II型TGF-β受体的腺病毒，以专门抑制TGF-β信号传导。在这些大鼠肝纤维化与对照组比较时，被壮观DMN治疗期间停止。其他研究已经证实，TGF-β抑制导致肝纤维化的发展9,10缓解。该模型也被用于在一个全局基因表达谱研究，以确定其中可以作为药物靶点抗纤维化疗法11其他纤维化标记物和蛋白质。
其他化学试剂用于诱导肝纤维化包括硫代乙酰胺（TAA）和c阿尔邦四氯化碳（CCL 4）。 TAA首次使用大鼠，后来在小鼠12。这种模式的优点包括：易于管理化学品的饮用水，以特色小结节性肝硬化生化变化模型的重复性。缺点包括：3个月肝纤维化的长时间周期来开发和缺乏对肝纤维化的诱导分子机制的理解。至于四氯化碳 ，它的使用有所下降，原因如下：它不模仿人类肝脏疾病，具有对臭氧层有害的影响，引起疼痛和痛苦动物，是非常对人体有毒，并要求其处理额外的预防措施和处置13,14。
长时间胆道梗阻（通过外科手术），作为肝硬化实验模型最早由Kountouras 等 15日报道。这种方法是无毒的人类和动物。 HH但是，开发所需的肝纤维化的时间变化。由马克斯等人 16 30份报告进行审查，发现花了由七天到四个星期后手术治疗肝纤维化的发展。所描述的病理变化模仿那些人慢性胆道纤维化和模型会更适合有意在这一领域的研究人员。
总之，DMN的大鼠在4周内恒定剂量的间歇给药产生肝纤维化模仿人类疾病。给药的大鼠显示肝损伤和肝纤维化实质的4,17逐步发展。疾病进展和严重性可以通过血样或处死动物在特定时间点进行监测和效果是高度可再现18。因此该模型已被广泛用于研究肝纤维化和肝硬化的机制，以及筛选潜在抗纤维化剂10,19,20。
Protocol
Representative Results
Discussion
我们已经描述了使肝纤维化的动物模型，并评估肝纤维化的严重性的方法。它提供DMN正确的剂量，坚持每周腹腔注射的日程安排是非常重要的。随着实验的进行，它是衡量老鼠和DMN注射每星期开始重新计算剂量的关键。请记住，DMN是有毒的，需要在通风柜进行处理。我们在通风柜进行腹腔注射为好。随着需要反复注射，注射的正确的克制和技术是重要的，可以避免污染物引入和内脏损害。我们很?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
The authors acknowledge the funding support from the Ministry of Education, Singapore, grant number MOE2010-IF-1-025.
Materials
| Dimethylnitrosamine | Wako | 147-03781 | |
| Formalin | Sinopharm chemicals | F63257009 | |
| Ethanol | Sigma | 64-17-5 | |
| Xylene | Fisher | 1330-20-7 | |
| Masson trichome stains | |||
| Aniline Blue | Electron Microscopy Sciences | #42755 | |
| Acid Fuschin | Electron Microscopy Sciences | RT42685 | |
| Scarlet Red | Electron Microscopy Sciences | #26905 | |
| Phosphotungstic Acid Hydrate | ALFA AESAR | ALFA40116.4 | |
| Phosphomolybdic Acid Hydrate | Sigma SG | 221856-25G | |
| Weigert's Iron Hematoxilyn | Merck | 1.15973.0002 | |
| DPX Mounting Medium | Merck | HX066873 | |
| Tissue processor | Leica | Leica TP 1020 | |
| Embedding machine | Sakura | Sakura Tissue Tek TEC5 Embedding System | |
| Microtome | Leica | Leica RM 2235 | |
| Vet Test Analyzer | Idexx | Vet Test 8008 |
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