颈动脉介入移植中颈静脉持久性转基因表达的家兔模型
Summary
该方法描述了介入静脉移植在家兔中的位置、移植物的转导以及持久性转基因表达的实现。这允许调查转基因及其蛋白产品在接枝静脉中的生理和病理作用, 以及对静脉移植病基因治疗的检测。
Abstract
静脉搭桥术是治疗闭塞性动脉疾病的常见方法;然而, 由于血栓形成、内膜增生和动脉粥样硬化的移植失败, 长期的成功是有限的。本文的目的是演示一种将双侧静脉介入移植在兔身上的方法, 然后用基因转移载体传感移植物, 达到持久的转基因表达。该方法可用于研究基因及其蛋白产物在正常静脉移植稳态中的生物学作用。它还允许测试转基因的活动, 以防止静脉移植失败, e., 转基因的表达是否能防止新生内膜的生长, 减少血管炎症, 或降低家兔动脉粥样硬化高脂肪饮食。在最初的生存手术中, 左颈外静脉的部分被切除, 并放置在双边上, 作为反向端对侧颈总动脉介入移植。在第二次生存手术中, 28 天后进行, 每一个移植从循环与血管片段分离, 流明是填补 (通过 arteriotomy) 与解决方案, 其中包含帮助者依赖腺病毒 (HDAd) 向量。经过20分钟的孵化, 矢量解吸气, arteriotomy 修复, 流恢复。这些静脉是在个别实验规程所规定的时间点上收获的。移植术与转导之间的28天延迟是确保移植静脉适应动脉循环的必要条件。这种适应避免了快速丢失的转基因表达, 发生在静脉移植转基因之前或紧接移植后。该方法具有在嫁接静脉中获得持久、稳定的转基因表达能力的独特性。与其他大型动物静脉移植模型相比, 家兔具有成本低、处理方便等优点。与啮齿动物静脉移植模型相比, 家兔具有更大、更易于操作的血管, 为分析提供了丰富的组织。
Introduction
动脉粥样硬化是一种慢性炎症性疾病, 其中脂质积累和炎症在血管壁导致血管腔狭窄, 心脏病发作, 中风, 四肢失去1,2。经皮介入治疗 (e., 血管成形术和支架植入术) 和医疗疗法 (g., 他汀类药物和抗血小板药物) 是治疗动脉粥样硬化的有效方法;然而, 在冠状动脉和外周循环中, 它们往往不能有效地治疗严重的梗阻性疾病。旁路移植术, 使用自体静脉段, 仍然是一个常见的程序治疗严重, 弥漫性冠状动脉和周围血管疾病3,4。然而, 在冠状动脉和外周循环中放置的静脉移植有很低的长期通畅率。在冠状动脉循环中, 大约10-20% 的静脉移植被闭塞1年, 50% 被闭塞10年5,6。在外周循环中, 静脉移植失败率为 30-50%, 5 年7。
基因治疗是一种很有吸引力的方法, 以防止静脉移植失败, 因为它可以提供一个治疗基因产品精确地在该疾病的地方。因此, 许多临床前研究已经测试了静脉移植基因治疗8,9。然而, 基本上所有这些研究已经检查的功效在早期点 (2-12 周)10,11,12,13,14,15,16,17. 我们知道没有证据表明基因治疗干预能提供持久 (多年) 保护, 防止晚期静脉移植失败, 通常是由新生内膜增生和动脉粥样硬化4引起的。我们开发了一种方法, 允许持久的转基因表达在嫁接静脉, 从而允许测试的基因治疗干预的后期以及早期的时间点。为实现持久的转基因表达, 该方法纳入 HDAd 载体和延迟转导策略。HDAd 载体提供长期的转基因表达, 因为他们缺乏病毒基因, 防止转基因细胞的识别 (和排斥) 的免疫系统18,19,20, 21. 延迟转导 (移植术后28天进行) 可防止移植22后早期发生的动脉过程中转基因细胞的丢失。
在静脉移植术中实现转基因表达的其他方法依赖于移植术10、11、12、15、16 时静脉移植的转导. ,17。在连续测量时, 使用这种方法的转基因表达在转导22、23后迅速下降。因此, 用这种方法进行的研究并没有对静脉移植后12周后的疗效进行检查, 最不评估疗效超过4周。相比之下, 我们的方法实现了静脉移植基因的表达, 稳定持续至少24周, 并根据类似的研究在动脉中进行-可能继续远长于22,24。我们知道没有其他静脉移植基因治疗干预, 达到稳定的转基因表达这一持续时间。
我们用兔子模型来发展我们的方法。另一些则使用啮齿动物、兔子或较大的动物来测试静脉移植基因治疗10、11、12、15、16、17、25、 26。与啮齿类动物模型相比, 兔子更昂贵, 并受到更严格的监管要求。然而, 与较大的动物相比(例如, 猪和狗), 兔子购买和房子的成本要低得多, 而且处理起来也更容易。此外, 兔血管与人体血管相似, 在生理上27, 它们足够大, 可以用来测试经皮介入治疗28,29, 他们提供了足够的组织,多个端点 (e., 组织学, 蛋白质, RNA) 可以使用单血管标本22,30检查。此外, 当带静脉移植的兔以高脂肪饮食喂养时, 他们发展静脉移植动脉粥样硬化31,32, 这是冠状动脉旁路移植失败的常见原因4,5.这些动脉粥样硬化兔静脉移植可以作为一个基质, 以测试的基因治疗干预提供这种方法。所提供的协议可以帮助研究者掌握在兔静脉移植中实现持久转基因表达所需的技术技能。
Protocol
所有动物协议和研究被华盛顿大学动物福利办公室批准。 1. 手术前 (所有手术) 麻醉兔与30毫克/千克氯胺酮和1.5 毫克/千克甲苯噻嗪通过肌肉注射 (IM) 在 paraspinous 肌肉。注意: 手术前不限制食物和水。 在等待足够深度的麻醉时, 在准备室和手术室 (或) 设置表。 准备准备房间剃须兔颈部和放置静脉 (IV) 口岸在耳朵 (仅生存手术)。放置眼科软膏, 芬太尼贴 (…
Representative Results
在操作员可以使用此方法生成实验数据之前, 必须验证新操作员的技术熟练程度。新的操作者必须达到的第一个里程碑是, 在初始静脉移植术和随后的延迟转导手术后, 保持静脉移植通畅。每次手术后90% 多的通畅是可取的和可实现的。可通过经皮超声评估无创通畅, 我们通常在术后5-7 天进行。在具有专利静脉的家兔中, 超声检查将显示一大血管, 在前颈部两侧有轻快的骶管?…
Discussion
本议定书的关键步骤包括麻醉的管理、抗凝、动脉/接枝静脉的手术操作以及接枝静脉的血流动力学测量。正确的麻醉管理在这一多项生存手术模型中是至关重要的, 包括两个相对较长的操作 (通常 3-3, 5 小时用于双侧静脉移植和 1.5-2.5 h 用于双边移植转导)。我们通过 nosecone 和气管插管进行麻醉, 发现插管改善了移植转导手术后的生存, 可能是因为正压通气预防肺不张和相关呼吸衰竭。家兔对插管有?…
Divulgations
The authors have nothing to disclose.
Materials
| Disposables | |||
| 3mL syringe with 24G needle | Becton Dickinson | 309571 | 2x for vein graft surgery; 2x for gene transfer surgery |
| 1 mL syringe with 27G needle | Becton Dickinson | 309623 | 2x for vein graft surgery, 5x for gene transfer surgery |
| 19G needle | Becton Dickinson | 305187 | Gene transfer surgery |
| 20 mL syringe, luer lock | Nipro Medical Corp | JD+20L | |
| Catheters, 24Ga x 3/4” | Terumo Medical Products | SROX2419V | |
| 21G needle | Becton Dickinson | 305165 | Gene transfer surgery and for 20 mL syringe of saline |
| Gauze 4” x 4” | Dynarex | 3242 | ~10-15 per surgery |
| 3-0 silk suture | Covidien Ltd. | S-244 | |
| 5-0 silk suture | Covidien Ltd. | S-182 | |
| 7-0 polypropylene suture | CP Medical | 8703P | Vein graft surgery |
| 7-0 polypropylene suture | CP Medical | 8648P | Gene transfer surgery |
| 5-0 polyglycolic acid suture | CP Medical | 421A | |
| 3-0 polyglycolic acid suture | CP Medical | 398A | |
| Alcohol swabs | Covidien Ltd. | 6818 | For the placement of I.V. line |
| Catheter plug | Vetoquinol | 411498 | |
| Ketamine HCl, 100 mg/mL | Vedco Inc. | 5098916106 | |
| Xylazine, 100 mg/mL | Akorn Inc. | 4821 | |
| Lidocaine, 20 mg/mL | Pfizer | 409427702 | |
| Marcaine 0.5% | Pfizer | 409161050 | |
| Beuthanasia D-Special | Intervet Inc. | NDC 00061047305 | Harvest surgery only |
| Buprenorphine HCl, 0.3 mg/mL | Patterson Veterinary | 12496075705 | |
| Saline IV bag, 0.9% sodium chloride | Baxter | 2B1309 | |
| Heparin (5000 U/mL) | APP Pharmaceuticals | NDC 63323-047-10 | |
| Papaverine (3.5 mg/ml) | American Reagent Inc. | NDC 0517-4002-25 | Diluted from 30mg/mL stock; Use 1 mL maximum |
| Fentanyl patch, 25 mcg/h | Apotex Corp. | NDC 60505-7006-2 | |
| Isoflurane | Multiple vendors | Catalog number not available | |
| Viral vector | Gene transfer surgery only | ||
| Surgical Instruments | |||
| Metzenbaum needle holder 7" straight | Roboz | RS-7900 | |
| Operating scissors 6.5" straight blunt/blunt | Roboz | RS-6828 | |
| Needle holder /w suture scissors | Miltex | 8-14-IMC | |
| Castroviejo scissors | Roboz | RS-5658 | |
| Castroviejo needle holder, 5.75" straight with lock | Roboz | RS-6412 | |
| Stevens scissors 4.25" curved blunt/blunt | Roboz | RS-5943 | |
| Alm retractor 4" 4X4 5mm blunt prongs | Roboz | RS-6514 | 2x |
| Backhaus towel clamp 3.5" | Roboz | 4x | |
| Micro clip setting forceps 4.75" | Roboz | RS-6496 | |
| Micro vascular clips, 11 mm | Roboz | ||
| Surg-I-Loop | Scanlan International | 1001-81M | 5 cm length |
| Bonaccolto forceps, 4” (10 cm) long longitudinal serrations, cross serrated tip, 1.2mm tip width | Roboz | RS-5210 | |
| Dumont #3 forceps Inox tip size .17 × .10 mm | Roboz | RS-5042 | |
| Graefe forceps, 4” (10 cm) long serrated straight, 0.8 mm tip | Roboz | RS-5280 | |
| Halstead mosquito forceps, 5" straight, 1.3 mm tips | Roboz | RS-7110 | 2x |
| Halstead mosquito forceps, 5" curved, 1.3mm tips | Roboz | RS-7111 | |
| Jacobson mosquito forceps 5" curved extra delicate, 0.9 mm tips | Roboz | RS-7117 | |
| Kantrowitz forceps, 7.25" 90 degree delicate, 1.7 mm tips | Roboz | RS-7305 | |
| Tissue forceps 5", 1X2 teeth, 2 mm tip width | Roboz | RS-8162 | |
| Allis-Baby forceps, 12 cm, 4×5 teeth, 3 mm tip width | Fine Science Tools | 11092-12 | 2x |
| Adson forceps, 12 cm, serrated, straight | Fine Science Tools | 11006-12 | |
| Veterinary electrosurgery handpiece and electrode | MACAN Manufacturing | HPAC-1; R-F11 | |
| Surgical Suite Equipment | |||
| Circulating warm water blanket and pump | Multiple vendors | Catalog number not available | |
| Bair hugger warming unit | 3M | Model 505 | |
| IV infusion pump | Heska | Vet IV 2.2 | |
| Isoflurane vaporizer and scavenger | Multiple vendors | Catalog number not available | |
| Veterinary multi-parameter monitor | Surgivet | Surgivet Advisor | |
| Veterinary electrosurgery unit | MACAN Manufacturing | MV-9 | |
| Surgical microscope | D.F. Vasconcellos | M900 | 25X magnification for vein graft surgery; 16X magnification for gene transfer surgery |
References
- Libby, P., Bornfeldt, K. E., Tall, A. R. Atherosclerosis: Successes, Surprises, and Future Challenges. Circ Res. 118, 531-534 (2016).
- Fowkes, F. G., et al. Comparison of global estimates of prevalence and risk factors for peripheral artery disease in 2000 and 2010: a systematic review and analysis. Lancet. 382, 1329-1340 (2013).
- Mohr, F. W., et al. Coronary artery bypass graft surgery versus percutaneous coronary intervention in patients with three-vessel disease and left main coronary disease: 5-year follow-up of the randomised, clinical SYNTAX trial. Lancet. 381, 629-638 (2013).
- de Vries, M. R., Simons, K. H., Jukema, J. W., Braun, J., Quax, P. H. Vein graft failure: from pathophysiology to clinical outcomes. Nat Rev Cardiol. 13 (8), 451-470 (2016).
- Harskamp, R. E., Lopes, R. D., Baisden, C. E., de Winter, R. J., Alexander, J. H. Saphenous vein graft failure after coronary artery bypass surgery: pathophysiology, management, and future directions. Ann Surg. 257, 824-833 (2013).
- Sabik, J. F. Understanding saphenous vein graft patency. Circulation. 124 (3), 273-275 (2011).
- Owens, C. D., Ho, K. J., Conte, M. S. Lower extremity vein graft failure: a translational approach. Vasc Med. 13, 63-74 (2008).
- Robertson, K. E., McDonald, R. A., Oldroyd, K. G., Nicklin, S. A., Baker, A. H. Prevention of coronary in-stent restenosis and vein graft failure: does vascular gene therapy have a role. Pharmacol Ther. 136, 23-34 (2012).
- Yla-Herttuala, S., Baker, A. H. Cardiovascular Gene Therapy: Past, Present, and Future. Mol Ther. 25, 1095-1106 (2017).
- Schwartz, L. B., et al. Adenoviral-mediated gene transfer of a constitutively active form of the retinoblastoma gene product attenuates neointimal thickening in experimental vein grafts. J Vasc Surg. (5), 874-881 (1999).
- Eefting, D., et al. Local lentiviral short hairpin RNA silencing of CCR2 inhibits vein graft thickening in hypercholesterolemic apolipoprotein E3-Leiden mice. J Vasc Surg. 50, 152-160 (2009).
- Handa, M., et al. Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft. J Vasc Surg. 48 (6), 1566-1574 (2008).
- Kloppenburg, G. T., Grauls, G. E., Bruggeman, C. A., Stassen, F. R. Adenoviral activin A expression prevents vein graft intimal hyperplasia in a rat model. Interact Cardiov Th. 8, 31-34 (2009).
- Eefting, D., et al. A novel urokinase receptor-targeted inhibitor for plasmin and matrix metalloproteinases suppresses vein graft disease. Cardiovasc Res. 88, 367-375 (2010).
- Eichstaedt, H. C., et al. Gene transfer of COX-1 improves lumen size and blood flow in carotid bypass grafts. J Surg Res. 161, 162-167 (2010).
- Kritz, A. B., et al. In vivo modulation of Nogo-B attenuates neointima formation. Mol Ther. 16 (11), 1798-1804 (2008).
- Peroulis, M., et al. The role of ex-vivo gene therapy of vein grafts with Egr-1 decoy in the suppression of intimal hyperplasia. Eur J Vasc Endovasc. 40, 216-223 (2010).
- Kochanek, S., et al. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and b-galactosidase. Proc Natl Acad Sci U S A. 93, 5731-5736 (1996).
- Parks, R. J., et al. A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. P Natl Acad Sci USA. 93, 13565-13570 (1996).
- Chen, H. -. H., et al. Persistence in muscle of an adenoviral vector that lacks all viral genes. P Natl Acad Sci USA. 94, 1645-1650 (1997).
- Wen, S., Graf, S., Massey, P. G., Dichek, D. A. Improved vascular gene transfer with a helper-dependent adenoviral vector. Circulation. 110, 1484-1491 (2004).
- Du, L., Zhang, J., Clowes, A. W., Dichek, D. A. Efficient gene transfer and durable transgene expression in grafted rabbit veins. Hum Gene Ther. 26, 47-58 (2015).
- Channon, K. M., et al. Efficient adenoviral gene transfer to early venous bypass grafts: comparison with native vessels. Cardiovasc Res. 35, 505-513 (1997).
- Flynn, R., et al. Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis. Mol Ther. 19, 1833-1841 (2011).
- George, S. J., et al. Sustained Reduction of Vein Graft Neointima Formation by Ex Vivo TIMP-3 Gene Therapy. Circulation. 124, 135-142 (2011).
- Chiu-Pinheiro, C. K., et al. Gene transfer to coronary artery bypass conduits. Ann Thorac Surg. 74, 1161-1166 (2002).
- Byrom, M. J., Bannon, P. G., White, G. H., Ng, M. K. Animal models for the assessment of novel vascular conduits. J Vasc Surg. 52, 176-195 (2010).
- Ribichini, F., et al. Effects of oral prednisone after stenting in a rabbit model of established atherosclerosis. J Am Coll Cardiol. 50, 176-185 (2007).
- Langheinrich, A. C., et al. Quantification of in-stent restenosis parameters in rabbits by Micro-CT. Rofo. 177 (4), 501-506 (2005).
- Wacker, B. K., Dronadula, N., Zhang, J., Dichek, D. A. Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis. Arterioscler Thromb. 37, 316-327 (2017).
- Zwolak, R. M., Kirkman, T. R., Clowes, A. W. Atherosclerosis in rabbit vein grafts. Arteriosclerosis. 9, 374-379 (1989).
- Qiang, B., et al. Statin therapy prevents expansive remodeling in venous bypass grafts. Atherosclerosis. 223, 106-113 (2012).
- Casa, L. D. C., Ku, D. N. Thrombus Formation at High Shear Rates. Annu Rev Biomed Eng. 19, 415-433 (2017).
- Chen, C., Coyle, K. A., Hughes, J. D., Lumsden, A. B., Ku, D. N. Reduced blood flow accelerates intimal hyperplasia in endarterectomized canine arteries. Cardiovasc Surg. 5 (2), 161-168 (1997).
- Binns, R. L., Ku, D. N., Stewart, M. T., Ansley, J. P., Coyle, K. A. Optimal graft diameter: effect of wall shear stress on vascular healing. J Vasc Surg. 10, 326-337 (1989).
- Oka, K., Mullins, C. E., Kushwaha, R. S., Leen, A. M., Chan, L. Gene therapy for rhesus monkeys heterozygous for LDL receptor deficiency by balloon catheter hepatic delivery of helper-dependent adenoviral vector. Gene Ther. 22, 87-95 (2015).
- Miyake, T., et al. Prevention of neointimal formation after angioplasty using nuclear factor-kappaB decoy oligodeoxynucleotide-coated balloon catheter in rabbit model. Circ Cardiovasc Interv. 7, 787-796 (2014).
- Chorny, M., et al. Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors. FASEB J. 27, 2198-2206 (2013).
- Hoshino, K., et al. Three catheter-based strategies for cardiac delivery of therapeutic gelatin microspheres. Gene Ther. 13, 1320-1327 (2006).
- Nouri, F., Sadeghpour, H., Heidari, R., Dehshahri, A. Preparation, characterization, and transfection efficiency of low molecular weight polyethylenimine-based nanoparticles for delivery of the plasmid encoding CD200 gene. Int J Nanomed. , 5557-5569 (2017).
- Jia, S. F., et al. Eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector. Cancer Gene Ther. , 260-266 (2002).
- Morishita, R., et al. Intimal hyperplasia after vascular injury is inhibited by antisense cdk 2 kinase oligonucleotides. J Clin Invest. 93, 1458-1464 (1994).
Citer Cet Article
Bi, L., Wacker, B. K., Dichek, D. A. A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts. J. Vis. Exp. (139), e57231, doi:10.3791/57231 (2018).