Summary

一种新的手术方法气管内注射生物活性剂在胎鼠模型

Published: October 31, 2012
doi:

Summary

我们开发了一种新的手术方法气管内给药的生物活性剂的胎鼠。运送路线是更有效地针对胎儿比常用的羊膜腔内注射的小鼠肺组织。这个过程至今没有在小鼠模型中被描述。

Abstract

产前肺部给药的细胞,基因或药物制剂可以提供各种遗传性和获得性疾病的新的治疗策略的基础。除了从先天性或继承异常要求交付的基因,几个非继承围产期条件,其中短期基因表达或药理干预是足以达到治疗效果的长期表达,被认为是作为潜在的未来的适应症种方法。候选短期产前治疗疾病的应用可能是的暂时性新生儿缺陷的表面活性蛋白B引起新生儿呼吸窘迫综合征1,2或高氧损伤的新生儿肺3。永久的治疗矫正的候选疾病囊性纤维化(CF)4,表面活性剂的不足5和α1-抗胰蛋白酶缺乏症的遗传变异。

<P类=“j​​ove_content”>一般情况下,产前基因治疗的一个重要优势是能够开始在开发的早期治疗干预,甚至是在病人的临床表现之前,从而防止个人造成不可弥补的损害。此外,胎儿各器官的细胞增殖率增加相比,成人的器官,它可以让一个更有效的基因或干细胞进入胎儿体内转移。此外, 在子宫内的基因传递的个体的免疫系统时,进行不完全成熟。因此,异源细胞的移植或补充的非功能性的或缺失的蛋白质与一个正确的版本应该不会引起免疫致敏的细胞,载体或转基因产物,它最近已被证明是细胞和基因疗法的情况下,7

在本研究中,我们调查的潜在直接定位在小鼠胎儿的气管米ODEL。本程序是在使用在大型动物模型中,如兔,羊8,即使在临床的设置9,但迄今尚未在小鼠模型中之前进行。在研究潜在的胎儿基因治疗遗传性疾病,如CF,作为第一个证明了的概念,因为不同的转基因小鼠品系中,有据可查的胚胎发育和胎儿的发育,不那么严格的广泛使用的小鼠模型是非常有用的伦理规范,妊娠期短及大窝产仔数。

已经描述了不同的访问路线针对的胎儿鼠肺,包括羊膜腔内注射10-12,(超声引导)肺内注射13,14和静脉内给药到卵黄囊船只15,16或脐静脉17。我们的新型外科手术使研究人员能够直接注射剂的选择,允许进入胎儿小鼠气管为更有效地提供比现有技术18呼吸道。

Protocol

1。小鼠交配,以获得所需的妊娠阶段时间交配怀孕NMRI小鼠,使他们怀孕18天(E18)(总妊娠E19.5)的手术时间。前和手术后他们被安置在过滤器顶部笼,免费使用水和周星驰在正常的室温和正常的日光。 2。 (IT)胎儿气管内注射(图1) 首先提交怀孕NMRI小鼠全身麻醉与异氟醚1.5%O 2的混合物在1.5 L / min的。的异氟醚的水平取决于年龄和应?…

Discussion

关键步骤

  • 小鼠品系,我们选择了与NMRI小鼠,因为他们有丰富的幼崽(平均窝产仔数14.4±1.8,自己的数据),以及容忍的干预,并具有良好的母体特征。
  • 通过子宫壁和胎膜配售的钱袋是关键的一步,因为你只是想揭露胎儿的头和肩膀,否则重新定位,几乎是不可能的,而不会造成创伤。
  • 胎儿的头向后伸展的最佳位置正上方的气管切开是必要的,以这样可以避免大血管(颈静脉?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

,MC和AVDP是支持的博士后研究员在佛兰德(IWT-佛兰德),通过科学和技术创新促进研究所的补助金。 JT持有的一部分的时间临床研究奖学金(KOOR,)UZ Leuven的。 DV是一个博士后研究员,鲁汶大学的资助,DBOF/10/062的支持。 MMdC是一个博士后研究员,从Conselho国家PesquisaËDesenvolvimento(CNPq)和伊拉斯谟的赠款支持。研究是由IWT-佛兰德,由欧共体授予DIMI的(LSHB-CT-2005-512146)和体内分子成像研究组(IMIR)的鲁汶大学。我们要感谢詹姆斯·M·威尔逊创办的宾夕法尼亚大学矢量核心,他们的AAV6.2包装质粒的腺相关病毒载体生产的一种礼物。

Materials

Name of the reagent Company Catalogue number Comments (optional)
NMRI mice Janvier, Le Genest St Isle, France
Isoflurane Isoba, Intervet / Schering-Plough Animal Health, Milton Keynes, UK
Prolene 6-0 Ethicon, Groot Bijgaarden, Belgium
Vicryl 5-0 Ethicon, Groot Bijgaarden, Belgium
50 μl Hamilton Glass Syringe, Model 1710.5 TLLX SYR Hamilton, Reno, NV, USA 5495-20
30G sharp needle Hamilton, Reno, NV, USA 7762-03
2% xylocaine AstraZeneca, Zoetermeer, The Netherlands

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Cite This Article
Carlon, M. S., Toelen, J., da Cunha, M. M., Vidović, D., Van der Perren, A., Mayer, S., Sbragia, L., Nuyts, J., Himmelreich, U., Debyser, Z., Deprest, J. A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model. J. Vis. Exp. (68), e4219, doi:10.3791/4219 (2012).

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