ADSC表移植到扩展食管内镜黏膜下剥离术之后防止狭窄
1Assistance Publique-Hôpitaux de Paris,Université Paris Descartes Sorbonne Paris Cité, 2Department of Gastroenterology,Hôpital Européen Georges Pompidou, 3UMR-S970,Université Paris Descartes Sorbonne Paris Cité, 4Department of Pathology,Hôpital Européen Georges Pompidou, 5Department of Radiology,Hôpital Européen Georges Pompidou
Summary
这项研究报告内窥镜脂肪组织的基质细胞（ADSC）的成功的方法折叠移植食管狭窄预防猪模型的扩展内镜黏膜下剥离术（ESD）之后。
Abstract
在过去几年中，细胞的表结构刺激了再生医学的广泛兴趣，尤其是对重建手术程序。多样化的技术脂肪组织衍生的基质细胞（脂肪干细胞）与各种生物材料相结合的发展导致了多种类型的组织工程替代品，如骨，软骨，和从啮齿动物，猪或人脂肪干细胞的脂肪组织的结构。扩展食管内镜黏膜下剥离术（ESD）负责食管狭窄的形成。狭窄预防仍然是具有挑战性的，没有可用的有效的治疗方法。先前的研究报道了犬模型和人类中的粘膜细胞片移植的有效性。脂肪干细胞归因抗炎特性，局部免疫调节作用，血管新生诱导，分化能力为间充质和非间充质细胞谱系。这个原始的研究描述了TRA内镜一个ADSC组织工程构建的nsplantation防止食管狭窄的猪模型。在ADSC结构是由一纸支撑膜在彼此分层2同种异体ADSC表。将脂肪干细胞标记的PKH67荧光允许基于探针的共聚焦激光显微内镜（pCLE）监控。上移植当天，进行5厘米和已知诱导食管狭窄半周的ESD。动物立即用胃镜ADSC 4移植结构。温和应用的10分钟后，获得的ADSC构建体的完整的粘合。动物处死28天所有动物均成功移植。移植证实第3天以积极的pCLE的评价。相比于移植的动物，对照组动物出现严重狭窄，与主要的纤维化组织的发展，越来越频繁的消化道麻烦，减少体重增加。在我们的模型中，allogeni移植Ç脂肪干细胞，双细胞片组织，增强的ESD后成功并以较低的食管狭窄率密切相关。
Introduction
浅表性食管肿瘤的管理层已与新的内镜技术的发展变化。如今，内窥镜切除是一线治疗。事实上，它是具有较低的发病率和死亡率比等于肿瘤结果1，2，3一手术相关。内窥镜粘膜切除术（EMR）和内镜黏膜下切除术（ESD）是最广泛使用的技术。在扩展浅表肿瘤的情况下，静电放电是优选的。相比EMR，ESD允许整块切除，无论病灶大小和形状4，5，6。 ESD的主要延迟并发症是食道狭窄的形成，这通常发生一周和两周之间切除后。最近公布的研究表明，狭窄形成是相关的切除的大小。日本内镜学会建议避免ESD尺寸大于¾食管周长，因为它们是在病例的90％以上与狭窄发展相关联，并且负责严重馈送麻烦和生活质量严重恶化的要大。
食管狭窄的预防仍然是具有挑战性的。参与狭窄形成机制只知道部分。狭窄的地层仿佛来自两个不同的机制，该协会导致：（1）促炎症细胞招聘和（2）过度纤维化发展7。几个预防性治疗方法已被提出。然而，结果并不令人满意，有什么好处和严重的副作用8,9。最近，日本队，大木等人。 ，提出了自体口腔粘膜细胞的单层细胞片移植到esophageal疤痕。在ESD 10,11后立即进行移植。他们证明这种创新方法的有效性，首先在犬模型，然后在病人。
脂肪组织来源的基质细胞（脂肪干细胞）是有希望在再生医学。其在几个领域中的应用已表明有趣的结果，特别是在伤口愈合过程。 ADSC疗法提供了几个优点，因为细胞是容易分离的，并具有抗炎特性，局部免疫调节作用，血管新生诱导，分化能力为间充质和非间充质系12，13，14相关联。
在先前的研究中，我们的团队表现出双ADSC表镜下移植治疗食管狭窄上一页成效在猪模型15增强的ESD后ention。在这篇文章中，ADSC板结构和内窥镜移植技术的报告介绍。
Protocol
所有的动物都根据动物研究伦理委员会（法国农业部的指导方针）处理。该协议获得在巴黎第五大学授权的动物experimentations当地伦理委员会的批准（注册号MESR 2035.02;医学学部巴黎笛卡尔，法国巴黎）。 1. ADSC文化和标签获得一个私人机构证实脂肪干细胞。在37℃和5％的CO 2与含10％胎儿小牛血清和1％抗生素（青霉素和链霉素）的α极限必需培养基培养同种异体脂肪干细胞。 </l…
Representative Results
脂肪干细胞并获得ADSC片的过程的培养示于图1。 图2示出了移植物的结构，在其纸载体膜在彼此层叠2 ADSC片组成。脂肪干细胞以前与PKH67荧光团标记，以便与 pCLE 体内移植监控。 图3示出了延长的食管内窥镜黏膜下层剥离术的不同步骤，从而产生一个5厘米和半周食管疤痕。 图4示出的内窥镜移植过程。移植是成…
Discussion
在这种猪模型中，ADSC表移植在技术上是成功的，并允许细胞植入监测体内 pCLE评价。临床，内窥镜，放射性，和组织学评价证明增强的ESD后在食管狭窄预防的内窥镜ADSC片的有效性。
含有靛蓝胭脂红染料片的ADSC甘油溶液的内窥镜移植是在再生医学的创新方法。大木等。第一次描述了内镜移植过程。在他们的研究中，细胞表构建体组成层叠在聚偏二氟乙烯的薄片口…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This study was supported and funded by the Avenir Foundation (Fondation de l’Avenir, 255 rue de Vaugirard, 75719 Paris cedex 15, Paris, France). This study would never have been conducted without the precious help of the veterinary team of the Laboratory of Biosurgical Research from the Alain Carpentier Foundation.
Materials
| Transparent endoscopic cap Q180 compatible | Olympus Optical Co | ||
| GIF-Q180 gastroscope | Olympus Optical Co | ||
| Videoscope System Exera II | Olympus Optical Co | ||
| Injection needle 18 G | Olympus Optical Co | ||
| Electrosurgery unit ERBE ICC 350 | ERBE Technology | ||
| Indigo carmin 1% | Life | ||
| Endoscopic hybrid knife | Life | ||
| Minisonde Z pCLE green probe | Mauna Kea Technology | You must learn how tu use the probe. The manipulation could be difficult | |
| Fetal bovine serum | Sigma Aldrich | 12105C | |
| Trypsin | Sigma Aldrich | T146 | |
| Alpha minimum essential medium | Thermo Fisher | 22561-021 | |
| Phosphate-Buffered Salines | Thermo Fisher | 10010-023 | |
| PKH67 dye kit | Sigma Aldrich | Mini67-KT | |
| 12-well temperature responsive cell culture dish | Upcell Thermo Scientific | 174900 | Feel the weel with 4 mL standard medium culture 30 minutes before seeding cells |
| Esomeprazole 40 mg | Biogaran | ||
| Moprhine sulfate 50 mg/ mL | Lavoisier | ||
| Amoxicilline 1 g | Biogaran | ||
| Ketamine 250 mg/5 mL | Panpharma | ||
| Propofol 10 mg/ mL | Fresenius | ||
| Hydrophobic paper | Carrefour |
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Tags
ADSC-sheet TransplantationEsophageal Stricture PreventionExtended Endoscopic ResectionEsophageal Superficial NeoplasiaPorcine ModelEndoscopic ADSC Sheet TransplantationCell ExpansionTrypsinTemperature Responsive Cell CultureESDVideo GastroscopeElectrosurgery UnitSubmucosal LayerCircumferential IncisionSubmucosal Dissection
Citer Cet Article
Perrod, G., Pidial, L., Camilleri, S., Bellucci, A., Casanova, A., Viel, T., Tavitian, B., Cellier, C., Clément, O., Rahmi, G. ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection. J. Vis. Exp. (120), e55018, doi:10.3791/55018 (2017).