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Medicina

体内兔颈总动脉内皮的基因转移

Published: May 06, 2018
doi:
10.3791/56982
, ,
1Department of Medicine,University of Washington

Summary

该方法将转基因技术引入兔颈动脉内皮细胞。转基因的引入可以评估转基因产品在正常动脉或疾病模型中的生物学作用。该方法对测定 DNA 调控序列的活性也有很重要的意义。

Abstract

该方法的目的是将转基因技术引入到兔颈总动脉孤立段的内皮细胞中。该方法实现了局灶性内皮选择性转基因, 从而使研究者能够确定内皮表达转基因的生物学作用, 并定量测定大动脉 DNA 序列的体内转录活性。内皮细胞。该方法采用手术隔离兔颈总动脉和 arteriotomy, 将转基因表达的病毒载体传递到动脉腔内。在腔内的载体的短潜伏期, 与随后的流明内容的愿望, 足以实现有效和持久的基因在内皮细胞的表达, 没有可检测的转导或表达以外的孤立的动脉段。该方法允许对正常动脉和人体血管疾病模型中的转基因产品的生物活性进行评估, 同时避免通过将基因传递定向到其他站点 (e. g) 而引起的系统性影响。肝脏) 或通过另一种途径, 通过生殖线转基因向内皮细胞提供遗传构造。这种方法的应用受限于需要熟练的外科医生和麻醉, 设备齐全的手术室, 购买和养兔的费用, 以及在基因转移载体的建设和使用方面的专门知识的需要。该方法获得的结果包括: 与转基因有关的动脉结构、细胞构成、细胞外基质或血管功能的改变;增加或减少动脉炎症;血管细胞凋亡的变化;疾病的进展, 迟缓, 或退化, 如内膜增生或动脉粥样硬化。该方法还允许测量本机和合成 DNA 调控序列在内皮细胞中改变转基因表达的能力, 提供的结果包括: 转基因 mRNA 的水平、转基因蛋白的水平和转基因的水平。酶活性。

Introduction

该方法的目的是在兔颈总动脉内皮中引入一种转基因。引入转基因可以评估转基因产品在正常动脉和兔模型的人类动脉疾病的生物学作用。在疾病模型中过度表达转基因可以揭示转基因 (及其蛋白产品) 是否显示为治疗剂1,2,3,4。纳入转基因表达式卡带中的顺式调节元素, 可以评估这些元素在动脉内皮体内5,6中的活动。了解特定的顺行调节元素的活动, 可用于设计更主动的表达盒, 并探讨在大动脉内皮细胞的基因调节机制在体内7。

家兔是人类血管生理学和疾病各个方面的重要模型。家兔与人类共有许多血管特征。例如, 基线血液学值, 止血调节和血管纵向张力是相似的兔子和人类的8。兔血管疾病模型复制了许多人类疾病的关键特征, 包括: 动脉瘤 (类似的几何和流特征)9, 血管痉挛 (类似的反应血管内治疗)10,11, 和动脉粥样硬化 (内膜斑块具有类似的功能, 包括一个核心富含脂质, 巨噬细胞, 和平滑的肌肉单元在纤维帽)12,13。因此, 兔子模型已经发展为许多血管疾病, 如血栓, 血管痉挛, 动脉瘤, 糖尿病, 血管移植狭窄, 动脉粥样硬化8,13,14, 15,16。

对于研究人员在动物模型中选择血管生理学和疾病, 兔有几个优点。与啮齿类动物相比, 较大的家兔血管允许手术操作更容易, 血管内装置的使用, 以及大量的组织进行定量测量。兔子比啮齿目动物的 phylogenetically 更接近灵长类动物17, outbred 兔的遗传多样性更接近人类的遗传变异。遗传多样性对临床前研究特别重要, 因为它们的性质-目的是开发可应用于基因多样化的人类群体的治疗方法。与许多, 如果不是所有其他模型物种, 兔子基因是容易克隆或合成, 因为兔子基因组已测序高覆盖率 (7.48x) [http://rohsdb.cmb.usc.edu/GBshape/cgi-bin/hgGateway？db=oryCun2]。与其他大型动物模型 (如狗, 猪, 或绵羊) 相比, 兔子购买和房子相对便宜, 他们更容易繁殖和处理。家兔特定的血管疾病模型各有其自身的优势和不足, 作为人类疾病的模型, 超出了本手稿的范围8,12,18。研究人员应审查这些优点和缺点, 以确定是否兔子是回答特定实验问题的最佳模型。

将脱氧核糖核酸 (DNA) 调控序列引入内皮细胞在体内可以在复杂的生理环境中对这些序列的活动进行调查。体外对转染内皮细胞的研究有助于对 DNA 调控序列进行初步评估;但是, 当研究重复在体内5,19,20时, 组织培养模型中的表达水平有时不会重现。体外系统也可用于探索蛋白质信号和内皮生理学的基本途径以及培养的血管细胞之间的沟通;然而, 在体内系统6、20中, 最好研究由相邻血管细胞或免疫系统复杂群体影响的更复杂的通路或调节网络。本文介绍的方法提供了一个平台, 探讨在血管内皮细胞内的转基因表达调控, 无论是否有疾病。体内系统还允许对生理和病理细胞串扰进行调查, 并确定免疫系统对基因表达式6的调节作用。

细菌线转基因 (尤其是小鼠) 是一种将转基因表达定向到内皮细胞的替代方法。这种方法可以提供终生的转基因表达, 以特定的启动子或监管区域介导的内皮靶向21,22。然而, 转基因小鼠的世代是费时和昂贵的, 必须经常测试一些转基因线, 以确保转基因的靶向所需的细胞类型和取得适当的转基因表达水平, 和实验结果在小鼠系统可能是应变依赖性。具有内皮靶向转基因的小鼠转基因模型有许多优点: 没有必要对每一个实验动物进行手术, 以达到转基因, 实验鼠可以与许多其他可用的转基因小鼠一起繁殖。为了测试基因和表型的相互作用, 有广泛的选择抗体, 与小鼠的蛋白质反应, 促进表征的表型。然而, 通过细菌线将转基因定向到内皮细胞, 通常会导致整个血管的转基因表达,22使得很难确定转基因产品的作用地点。当转基因产品分泌时尤其如此, 因为在整个血管的血管内皮细胞分泌的转基因产品可能在动物体内的任意数量的部位有生物活性。虽然本手稿中描述的方法需要技术专长和专门设施, 但它的耗时和成本比开发内皮特异的转基因小鼠线要少得多。它允许在大动脉段的内皮细胞中有选择地评估蛋白质的功能, 并且它允许使用对侧的颈动脉作为配对的控制 (消除系统因素, 可以改变在实验动物-例如, 血压或胆固醇水平-作为不受控制的变量)。

基因疗法是治疗血管疾病, 特别是慢性病的一种有希望的方法, 因为单一的应用可以提供持续或可能终生表达的治疗基因23。基因治疗在体细胞基因转移动物模型中的治疗前景已被探索, 通常针对肝脏24,25, 这是一个相对容易的目标, 因为许多血液传播的病毒载体是 hepatotropic 的。然而, 为了对血管疾病产生影响, 针对肝脏的基因治疗必须实现蛋白质的系统性过度表达。这通常需要大剂量的向量, 这可能是有毒的, 甚至是致命的26。此外, 蛋白质系统水平的提高增加了非靶向副作用的风险, 这可能使实验结果的解释复杂化甚至模糊不清。本手稿所描述的针对血管内皮的局部基因治疗可以避免系统性的副作用, 因为注入的载体没有广泛传播到转基因动脉段之外, 而且可以实现局部血管效应, 而无需系统血浆蛋白水平的变化。27此外, 传感器动脉段所需的载体数量远低于实现强健肝传导的需要。随着时间的推移, 来自肝脏的转基因表达被报告为下降, 可能是由于细胞更替, 如果要维持高水平的转基因表达, 需要反复用药。28相比之下, 内皮细胞的低更替率为喂食家兔至少48周提供稳定表达, 在胆固醇喂养家兔的动脉粥样硬化性病变中至少有24周。1,27

为了确定这种基因转移到兔颈总动脉内皮的方法是否合适, 应在特定研究目标的背景下考虑其优缺点 (表 1)。该方法的优点包括: outbred 兔比近交系小鼠更好地代表人类遗传多样性 (对前期工作很重要);兔子提供更大的血管, 便于操作和更多的组织进行分析;该方法能较快地实现转基因小鼠的血管内皮靶向转基因表达;矢量剂量可以很容易地调整为模型可变水平的转基因表达;可对大动脉内皮的特定过程进行研究;局部血管转基因允许同一动物的相对颈动脉作为一种控制, 将系统因素排除为不受控制的变量。缺点包括: 需要特殊设施和专门知识;更少的基因改良的背景, 以实验是在兔子比在小鼠;对兔子和老鼠蛋白的抗体的选择较少 (对于转基因蛋白和其他抗原的 immunodetection, 这在解释实验结果中可能是重要的)。

Protocol

这里描述的所有方法都是由华盛顿大学动物福利和相关机构动物护理和使用委员会 (IACUC) 批准的, 并已按照和遵守所有相关的管理和机构指引。 注: 兔颈总动脉的基因转移是由外科医生在麻醉师或助手的帮助下进行的。 1. 基因转移到兔颈总动脉: 手术前 麻醉兔子。给兔子称重。将30毫克/千克氯胺酮和2毫克/千克甲苯噻嗪组合成注射器。注射氯胺酮/甲苯噻嗪肌 (IM) 在 paraspinous 肌肉…

Representative Results

为了实现这种方法的信心, 初步实验是必要的, 以确定该操作者获得有效和可再生的基因转移, 以转基因表达主要在腔内内皮细胞。根据我们的经验, 这是最容易评估使用的向量, 表达β-乳糖酶。5-溴-4-氯-3-哚β D-galactopyranoside (X-加仑) 染色的颈总段清除后3天的载体输液, 以及测量β-乳糖酶 mRNA (信使 RNA) 与定量反向转录聚合酶链反应 (qRT PCR), 将揭示效率, 重现性和转基因细胞的…

Discussion

外科技术的某些方面值得特别注意。通过仔细解剖, 充分暴露和动员颈总动脉, 将促进基因转移和 arteriotomy 修复。然而, 在解剖过程中, 应尽量减少对颈动脉的直接操纵, 以防止血管痉挛。此外, 在动脉旁的任何出血应以纱布轻压停止, 淤血血液应立即清除, 用生理盐水冲洗区域。同样重要的是避免损害迷走神经, 这是平行于颈总动脉。修剪计划 arteriotomy 区域中的膜将有助于操作人员执行干净和功能 a…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢 AdVec, 公司允许使用 HDAd 试剂, 朱莉娅 Feyk 为行政协助, 和比较医学兽医服务部的外科咨询和支持。这项工作得到了 HL114541 和小约翰. 洛克慈善信托基金的支持。

Materials

Disposables
3mL syringe with 24G needle Becton Dickinson 309571 2x for gene transfer surgery; 3x for harvest surgery
1mL syringe with 27G needle Becton Dickinson 309623 6x for gene transfer surgery; 1x for harvest surgery
20mL syringe, luer lock Nipro Medical Corp JD+20L
Catheters, 24G x 3/4" Terumo Medical Products SROX2419V
19G needle Becton Dickinson 305187 Gene transfer surgery only
21G needle Becton Dickinson 305165 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 Gene transfer surgery only
7-0 polypropylene suture CP Medical 8648P Gene transfer surgery only
5-0 polyglycolic acid suture CP Medical 421A Gene transfer surgery only
3-0 polyglycolic acid suture CP Medical 398A Gene transfer surgery only
Alcohol swabs Covidien Ltd. 6818 For placement of I.V. line
Catheter plug Vetoquinol 411498 Gene transfer surgery only
Ketamine HCl, 100 mg/mL Vedco Inc. 05098916106
Xylazine, 100 mg/mL Akorn Inc. 4821
Lidocaine HCl, 2% Pfizer 00409427702
Bupivacaine HCl, 0.5% Pfizer 00409161050
Beuthanasia D-Special Intervet Inc. NDC 00061047305 Harvest surgery only
Buprenorphine HCl, 0.3 mg/mL  Patterson Veterinary 12496075705 Gene transfer surgery only
Saline IV bag, 0.9% sodium chloride Baxter 2B1309 2x for gene transfer surgery; can use vial of sterile saline in place of one
Heparin  (5000 U/mL) APP Pharmaceuticals NDC 63323-047-10 Gene transfer surgery only
Fentanyl patch, 25 mcg/hr  Apotex Corp. NDC 60505-7006-2 Gene transfer surgery only
Isoflurane Multiple vendors Catalog number not available
Gene transfer vector Dilute 350 µL per artery; 2 x 1011 vp/mL for adenovirus; gene transfer surgery only
Surgical Instruments
Metzenbaum needle holder 7" straight Roboz RS-7900 Gene transfer surgery only
Operating scissors 6.5" straight blunt/blunt Roboz RS-6828
Needle holder /w suture scissors Miltex 8-14-IMC Gene transfer surgery only
Castroviejo scissors Roboz RS-5658
Castroviejo needle holder, 5.75" straight with lock Roboz RS-6412 Gene transfer surgery only
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 Gene transfer surgery only
Micro vascular clips, 11 mm Roboz 2x for gene transfer surgery only
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 X .10mm Roboz RS-5042
Graefe forceps, 4” (10 cm) long serrated straight, 0.8mm tip Roboz RS-5280
Halstead mosquito forceps,  5" straight, 1.3mm 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
Forced air warming unit 3M Bair Hugger Model 505 Gene transfer surgery only
IV infusion pump Heska Vet IV 2.2 Gene transfer surgery only
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 Needs ~16x magnification
DOWNLOAD MATERIALS LIST

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Tags

In Vivo Gene TransferRabbit Common Carotid ArteryEndotheliumVascular BiologyGene TherapyTransgene ExpressionSurgical ProcedureDissectionCarotid ArteryLigationMicroscope

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Wacker, B. K., Bi, L., Dichek, D. A. In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium. J. Vis. Exp. (135), e56982, doi:10.3791/56982 (2018).
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