TNBS诱导的啮齿动物模型研究机械应力在克罗恩病中的致病作用
Summary
本方案描述了啮齿动物中克罗恩病样结肠炎模型的发展。透壁炎症导致TNBS滴注部位狭窄，并且在狭窄近端的节段观察到机械性肿大。这些变化允许研究结肠炎的机械应力。
Abstract
炎症性肠病（IBD）如克罗恩病（CD）是胃肠道的慢性炎症性疾病，在欧洲和美国影响约20/1，00，000。克罗恩病的特征是透壁炎症、肠纤维化和腔内狭窄。虽然抗炎疗法可能有助于控制炎症，但它们对克罗恩病的纤维化和狭窄没有疗效。克罗恩病的发病机制尚不清楚。目前的研究主要集中在描绘失调的肠道免疫反应机制上。虽然CD相关的透壁炎症，肠纤维化和腔内狭窄都代表对肠壁的机械应力，但机械应力在CD中的作用尚未明确定义。为了确定机械应力在克罗恩病中是否起独立的致病作用，已经开发了TNBS诱导的啮齿动物CD样结肠炎模型方案。这种TNBS诱导的透壁炎症和纤维化模型类似于结肠中CD的病理特征。它是由结肠内将TNBS滴注到成年Sprague-Dawley大鼠的远端结肠中诱导的。在该模型中，透壁炎症导致TNBS滴注部位（站点I）狭窄。在滴注部位近端（P位点）的部位观察到机械性膨胀，代表机械应力但不可见炎症。炎症远端的结肠部分（D位点）既不发炎也不出现机械应激。在不同位点（P，I和D）观察到基因表达，免疫反应，纤维化和平滑肌生长的独特变化，突出了机械应力的深远影响。因此，这种CD样结肠炎模型将帮助我们更好地了解CD的致病机制，特别是机械应力和机械应力诱导的基因表达在CD免疫失调，肠纤维化和组织重塑中的作用。
Introduction
炎症性肠病（IBD），包括溃疡性结肠炎（UC）和克罗恩病（CD），其特征在于胃肠道（GI）的慢性炎症。它影响约1-2百万美国人1。据估计，美国IBD治疗的年度成本为118亿美元。与溃疡性结肠炎不同，克罗恩病的特征是透壁性炎症和狭窄形成2，3。狭窄形成（狭窄）见于高达 70% 的克罗恩病患者3 ，可能由透壁炎症（炎性狭窄）或肠纤维化（纤维化狭窄）引起 4，5。肠纤维化的特征在于过度的胶原沉积和其他细胞外基质（ECM），平滑肌细胞（SMC）是参与该过程的主要间充质细胞类型之一3，4。与肥大相关的平滑肌增生是 CD6 中纤维化狭窄的另一个显著组织学变化。虽然克罗恩病患者的狭窄形成与慢性炎症有关，但除手术治疗外，没有有效的抗炎治疗2，6。然而，手术后复发率几乎为100%，给予足够的时间2，7。作为炎症反应，纤维化和SMC增生也可能在肠道中的非炎症性疾病（即肠梗阻）中发展8，9;据信，炎症依赖性和独立机制都与狭窄形成有关3，4。鉴于对炎症依赖性机制的广泛研究尚未转化为任何有效的狭窄形成疗法，因此需要研究炎症非依赖性机制在肠纤维化中的可能作用。
作为一种非炎症因子，与水肿、炎性细胞浸润、组织变形、纤维化和狭窄10，11，12，13 相关的机械应力 （MS） 常见于 IBD，尤其是以透壁炎症为特征的 CD。机械应激在狭窄性克罗恩病中最为显著，其中炎症部位的狭窄（炎症性或纤维化）在局部组织中表现出机械应力，并导致梗阻部位近端的管腔扩张10，14。先前的体外研究表明，机械应激会改变胃肠道组织中特定炎症介质（即COX-2，IL-6）8，14，15和生长因子（即TGF-β）的基因表达，特别是肠道平滑肌细胞（SMC）16。最近的研究还发现，特定的促纤维化介质如结缔组织生长因子（CTGF）的表达对机械应力高度敏感17，18。据推测，机械应力可能在CD相关炎症，纤维化和组织重塑中起独立的致病作用。然而，机械应力在克罗恩病肠道炎症、纤维化和平滑肌增生中的致病意义在很大程度上仍未得到探索。这可能部分是因为炎症是一个比机械应力更明显，研究得更好的过程。更重要的是，还没有明确定义的IBD动物模型来区分机械应力和炎症的影响。
本研究描述了一种啮齿动物模型，该模型由结肠内注射半抗原试剂2，4，6-三硝基苯磺酸（TNBS）19，20诱导的克罗恩病样结肠炎，可用于研究机械应力在CD中的作用。发现TNBS滴注诱导远端结肠局部（长度约2cm）透壁炎症，伴有管腔狭窄（狭窄）。狭窄导致明显的肠胀（机械应力）14，15 但在滴注部位近端的结肠节段不可见炎症。相反，狭窄部位远端的结肠节段既没有炎症也没有机械应力。在三个不同的位点观察到基因表达，炎症，纤维化和SMC增生的显着位点特异性变化。结果表明，机械应力，特别是机械应力诱导的基因表达，可能在克罗恩结肠炎的纤维化和增生中起关键作用。
Protocol
Representative Results
Discussion
TNBS诱导的结肠炎于1989年引入，自那时以来一直被用作克罗恩病的实验模型19，20，23。该模型在啮齿动物中的显着特征包括发生透壁炎症，其与人类克罗恩病19，20中发生的组织病理学病变非常相似。以前对该模型的研究主要集中在可见炎症部位（I位点）的粘膜层中的异常免疫反应<su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了NIH（R01 DK124611至XZS）和美国国防部（W81XWH-20-1-0681至XZS）的资助。组织学工作是在UTMB外科病理学实验室的帮助下完成的。
Materials
| ACT-1 Control Software Ver2.63 | Nikon | DXM1200F | |
| C1000 Touch Thermal Cycler with 96-Well Fast Reaction Module | BIO-RAD | 1851196 | |
| CFX96 Optical Reaction Module for Real-Time PCR Systems | BIO-RAD | 1845097 | |
| Dako Agilent Artisan Link Pro Special stainer | Dako | AR310 | |
| Dako-Agilent Masson's Trichrome Kit ref# AR173 | Dako | AR173 | |
| DXM1200 Digital Color HR Camera | Nikon | DXM1200 | |
| Eukaryotic 18S rRNA Endogenous Control | ThermoFisher Scientific | 4352930E | |
| E-Z Anesthesia | E-Z Systems Inc. | EZ-155 | |
| GraphPad Prism 9 | GraphPad | 9.0.2 (161) | |
| Hard-Shell 96-Well PCR Plates, low profile, thin wall, skirted, white/clear | BIO-RAD | HSP9601 | |
| HBSS (Corning Hank's Balanced Salt Solution, 1x without calcium and magnesium) | CORNING | 21-021-CV | |
| HM 325 Microtome | Thermo Scientific | 23-900-667 | |
| Isoflurane | Piramal | NDC 66794-017-10 | |
| LI-COR Odyssey Digital Imaging System | LI-COR | 9120 | |
| Mastercycler epGradient Thermal Cycler with Control Panel 5340 Thermal Cycler | Eppendorf | 5341 | |
| Medical grade open end polyurethane catheter | Covidien | 8890703013 | |
| NanoDrop 2000/2000c Spectrophotometers | Thermo Fisher Scientific | ND2000CLAPTOP | |
| Nikon Eclipse E800 Upright Microscope | Nikon | E800 | |
| Nitrocellulose/Filter Paper Sandwiches Pkg of 50, 0.45 μm, 7 x 8.5 cm | BIO-RAD | 1620215 | |
| Polyethylene Glycol 3350, Osmotic Laxative | Miralax | C8175 | Dose: 17g in 226 mL of water |
| RNeasy Mini Kit (250) 250 RNeasy Mini Spin Columns, Collection Tubes (1.5 mL and 2 mL), RNase-free Reagents and Buffers |
QIAGEN | 74106 | |
| SuperScript III First-Strand Synthesis System | ThermoFisher Scientific | 18080051 | |
| TaqMan Gene Expression Assays Rn00573960_g1 CTGF Probe | ThermoFisher Scientific | 4331182 | |
| TaqMan Gene Expression Assays Rn99999011_m1 IL6 Probe | ThermoFisher Scientific | 4331182 | |
| TaqMan Fast Advanced Master Mix | ThermoFisher Scientific | 4444557 | |
| Tissue-Tek Prisma H&E Stain Kit #1 | Sakura | 6190 | |
| Tissue-Tek Prisma Plus Automated Slide Stainer | Sakura | 6171 | |
| TNBS (Picrylsulfonic acid solution) | SIGMA-ALDRICH | 92822 |
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