的同种免疫引起的血管性排斥反应和移植物动脉硬化小鼠模型
Summary
We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.
Abstract
Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.
Introduction
在过去的30多年，在免疫抑制药物的进步已经减少移植排斥反应所致的急性排斥反应，但慢性排斥反应仍然是一个主要挑战。慢性心脏移植排斥反应的主要表现是移植动脉硬化（TA）1,2。这种情况的特点由内膜增生和移植动脉血管舒缩功能障碍和发展为内皮细胞和平滑肌细胞的接受者免疫系统的免疫学定位的结果。移植物血管因承认外国肽主要组织相容性复合体（MHC）的具体目标是通过TA的发展，同时保留主船3只强调在移植动脉。为了保持这是TA不发生实验时，收件人是基因完全相同的供体或当收件人缺乏T细胞和B细胞4观察。免疫介导的血管损伤及dysfunctioÑ 引起内膜增厚和纤维化的发展，以及脂质和ECM蛋白的异常积累，在TA 5。内膜增厚趋于整个动脉树4-6同心。移植物丢失和死亡通常发生从同种异体移植物的动脉4的管腔闭塞导致渐进缺血的结果。
1991年，Mennander 等 7率先主动脉干预模型大鼠模型TA。一些研究小组已经适应随后此过程中使用的小鼠。在此模型中，同种异体移植主动脉段开发病灶具有特征媲美的TA在临床移植观察。这包括内膜增厚特征在于平滑肌细胞样细胞和受体白细胞7的积聚。在过去的二十年中该模型已被用于产生重要的洞察的血管损伤，排斥和TA的机制。它可以是我们编辑审查过程中动脉病理与免疫和血管反应的问题。抗原错配的影响选择适当解决这些问题的能力。
跨越完整MHC障碍移植允许已知参与器官移植排斥反应的免疫反应的全面评价。这包括直接CD4和CD8 T细胞识别和外国肽-MHC的靶向通过接枝衍生细胞呈现，间接的CD4（以及可能的CD8）的T细胞识别和由收件人抗原呈现接枝衍生同种异体抗原呈递细胞靶向和抗体同种抗原对血管细胞表面介导的8认可。但是，为了在完全MHC错配的实验损伤的血管反应可以比所观察到临床上不同。约翰逊等人 9表明，在整个完整的MHC错配屏障移植主动脉插入移植物，大部分内膜细胞受体的起源和不供体来源。这比在人类移植大多数内膜平滑肌细胞是供体来源9,10的观察不同。考虑到这一限制，已经开发了替代实验模型是涉及跨越次要组织相容性抗原错配接枝触发血管反应更接近于那些在临床移植11观察。虽然这些替代模式允许重要的结论可以做出关于驱动的TA，这引起血管排斥次要组织相容性抗原不匹配的移植物不完全重新投降那些发生在临床免疫进程的发展的血管反应。例如，次要组织相容性抗原是由接枝反应的抗体12差识别。鉴于上述考虑，要考虑的病理questi是非常重要的上时，选择在主动脉插入模型中使用的抗原不匹配的类型检查。在这里，我们描述了小鼠动脉介入移植了详细的方案。我们描述完整MHC错配的小鼠，但相同的协议被用于在其他抗原错配的小鼠品系接枝之间插入嫁接。
Protocol
Representative Results
Discussion
我们已经描述了一个协议，用于主动脉插入接枝小鼠是用于研究免疫介导的血管性排斥和TA有用。该模型可用于研究的TA的原因以及新的治疗策略的发展。它已被用于在过去，建立在TA 14,17-21适应性免疫，细胞毒性T细胞应答，细胞因子介导的CD4 T细胞效应的反应，和抗体介导的移植物损伤的重要作用。在小鼠中动脉移植是困难的，因为该动物的明显小尺寸;然而，随着实践和尽职调查，成功?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由健康研究和心脏及中风BC与育空地区的基金会（JCC）的加拿大学院资助。
Materials
| Name | Company | Catalogue | Comments |
| C57BL/6J (H-2b) | Jackson Laboratories, Bar Harbour ME | Strain# 000664 | |
| Balb/cBYJ | Jackson Laboratories, Bar Harbour ME | Strain# 001026 | |
| Ketamine Hydrochloride Injection USP 100 mg/ mL | Ketalean | DIN 00612316 | |
| Xylazine Injection 20 mg/mL | Rompum | DIN 02169592 | |
| Ketoprofen Injection 100 mg/mL | Anafen | DIN 01938126 | |
| Butorphanol Tartrate injection 10 mg/mL | Torbugesic | DIN 008450000 | |
| Buprenorphine Injection 0.3 mg/mL | Reckitt Benckiser | B.N. 5241 | |
| Atipamezole hydrochloride sterile injectable solution | Antisedan | DIN 02237744 | |
| Heparin Sodium Injection, USP, 1000 units/mL | McKesson Distribution | DIN 02264315 | |
| Tears naturale ophthalmic ointment | Alcon | DIN 02082519 | |
| Stereomicroscope | Leica | M80 | |
| 0.9% Sodium Chloride, sterile | Baxter Corporation | ||
| Lactated Ringer’s solution, sterile | Baxter Corporation | ||
| 0.9% Sodium Chloride Injection, sterile, 10 mL | Baxter Corporation | ||
| Alcohol Prep Pads | Loris | ||
| Povidone Iodine | Betadine | ||
| Chlorohexidine Gluconate 4% w/v | Germi-Stat | ||
| Black Polyamide Monofilament | AROSurgical Instruments | T4A10Q07 | |
| Suture, 10-0 suture, 70 microns | Corporation | ||
| Blue monofilament suture 5-0, P3 needle | Ethicon | 8698G | |
| 1 ml Syringe | BD | REF 309659 | |
| 10 ml Syringe | BD | REF 309604 | |
| 1cc TB insulin syringe with 28G 1/2 | BD | REF 309309 | |
| 25G 7/8, hypodermic needle | BD | REF 305124 | |
| 27G 1/2, hypodermic needle | BD | REF 305109 | |
| Colibri Retractor- 1.5cm spread 4cm | Fine Science Tools | 17000-04 | |
| S&T CAF-4 Clip applying forceps, without lock | Fine Science Tools | 00072-14 | |
| Supergrip forceps, S&T | Fine Science Tools | 00632-11 | |
| Medical No.5 forceps | Fine Science Tools | 11253-20 | |
| Lexer Baby Scissors | Fine Science Tools | 14078-10 | |
| Micro Adson forceps serrated | Fine Science Tools | 11018-12 | |
| Vannas-Tubingen microscissors | Fine Science Tools | 15003-08 | |
| Micro clamps, b-1; 3.5mm x 1mm; 7mm length | Fine Science Tools | 00396-01 | |
| Graefe-forceps, 10cm 1×2 teeth | Fine Science Tools | 11054-10 | |
| Castroviejo with lock and tungsten jaws | Fine Science Tools | 12565-14 | |
| Hot glass bead sterilizer | Inotech 250 | IS-250 – Steri-250 | |
| Non-woven gauzes | Progene | ||
| Cotton Tipped Applicators | Puritan | ||
| Beard Trimmer | Wahl | ||
| Heating pad | Sunbeam |
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