增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型
Summary
在这里, 我们提出了一个方案, 研究增殖性糖尿病视网膜病变的病理生理学使用患者衍生的, 手术切除, 纤维血管组织的三维本地组织表征和体外培养。这种体外培养模型也适合测试或开发新的治疗方法。
Abstract
糖尿病视网膜病变 (dr) 是糖尿病最常见的微血管并发症, 也是工作年龄成年人失明的主要原因之一。目前没有糖尿病和氧引起的视网膜病变的动物模型会出现人类增殖性糖尿病视网膜病变 (pdr) 所表现出的全系列渐进性变化。因此, 对疾病发病机制和病理生理学的了解在很大程度上依赖于使用组织学切片和玻璃体样本的方法, 这些方法只能提供有关致病因素的稳态信息。越来越多的证据表明, 在三维 (3d) 微环境中的动态细胞和细胞外基质 (ecm) 相互作用对于新的治疗策略。因此, 我们假设用 pdr 手术切除眼睛的病理性纤维血管组织可以可靠地揭示这种毁灭性疾病的细胞和分子机制, 并测试新的临床潜力干预。为此, 我们开发了一种新的方法, 用于手术切除患者衍生的纤维血管组织 (ft) 的三维体外培养, 该方法将作为人类 pdr 病理生理学的相关模型。将 ft 分解成外植体, 并嵌入纤维蛋白基质中, 用于体外培养和三维表征。通过对原生 fts 和终点培养物的全安装免疫荧光进行彻底的研究, 从而可以对组织组成和多细胞过程进行彻底的研究, 突出了3d 组织级表征对发现相关特征的重要性。pdr 病理生理学。该模型将允许在 pdr 组织结构和微环境中动态生化和物理相互作用的复杂背景下同时评估分子机制、纤维素组织过程和治疗反应。由于该模型重述了 pdr 病理生理学, 因此也可进行测试或开发新的治疗方法。
Introduction
dr 是糖尿病的一种严重的眼部并发症, 在过去30年中, 这种疾病已达到巨大的比例1。诊断20年后, 几乎每个1型糖尿病患者和60% 的2型糖尿病患者都有视网膜病变的迹象, 使糖尿病本身成为正常工作年龄失明的主要原因之一.根据微血管变性和缺血损伤的程度, dr 分为非增殖 dr (非 pdr) 和增殖 dr (pdr)。终末期疾病 pdr 的特点是缺血和炎症引起的新生血管和纤维化反应在玻璃体视网膜界面。在未经治疗的情况下, 这些过程将导致失明, 由于玻璃体出血, 视网膜纤维化, 视网膜三度脱离, 和新生血管性青光眼3,4。尽管最近的进展, 目前的治疗方案只针对 dr 阶段, 包括糖尿病黄斑水肿和 pdr, 当视网膜损伤已经接踵而至。此外, 很大一部分 dr 患者没有从目前的治疗水族馆中受益, 这表明迫切需要改进治疗 4、5、6.
到目前为止, 已经开发出多个其他体内疾病发展模型和糖尿病动物模型, 但没有一个模型可以概括人类 pdr7,8中观察到的全部病理特征。此外, 越来越多的证据表明, 治疗反应与 ecm 组合以及细胞和无细胞微环境之间的空间排列和相互作用密切相关。因此, 我们开始开发一个临床相关的模型的人 pdr 利用 ft 病理材料, 通常是从眼睛接受玻璃体切除术作为 pdr10的手术管理的一部分。
这份手稿描述了3d 体外培养和定性的手术切除, pdr 患者衍生病理 ft 的协议。这里描述的方法已被用于最近的一份出版物, 证明了成功解构本地 3d pdr 组织景观, 并重述了 pdr 病理生理学的特征, 包括血管生成和纤维化反应的异常血管结构11。该模型还揭示了从薄薄的组织学切片中不易被欣赏的新特征, 如空间限制的凋亡和增殖以及血管胰岛形成11。玻璃体液已被其他人成功地应用于三维内皮球体培养物, 以评估其血管生成潜力和血管静止分子12的有效性。当与体外三维淋巴细胞内皮细胞 (lec) 球体萌发试验结合使用 pdr 玻璃体作为刺激物时, 我们的模型揭示了可溶性玻璃体因子以及新生血管组织中的局部线索对作为刺激物的贡献。但对 lec 参与 pdr 病理生理学的情况了解甚少,3, 11。在 pdr 的管理中, 玻璃体视网膜手术是一个例行执行但具有挑战性的程序。由于外科器械和技术正在看到不断的进步和成熟, 及时和保守地去除纤维血管增生标本不仅提高视力, 而且还提供了宝贵的组织材料研究人类组织微环境复杂翻译方面的 pdr 病理生理学和治疗反应。
Protocol
这项研究得到了赫尔辛基大学医院机构审查委员会和伦理委员会的批准。每个病人都得到了签字的知情同意。 1. 解决方案、介质和设备的准备 在收集纤维血管组织 (ft) 之前准备以下设备, 以确保快速处理。 灭菌高压灭菌器两个微解剖推子。 在900毫升去离子水中溶解1个预称的 pbs 片 (0.14 m ncl, 0.0027 m kcl, 0.010 m po 4-3), 制备 1x磷酸盐缓冲盐水…
Representative Results
对 pdr 纤维血管组织特性和蛋白表达的深入了解主要依靠玻璃体样品和薄组织学 ft部分 3,15,16,17。为了建立一种方法, 彻底研究三维组织组织和多细胞生理病理过程的 pdr, 我们提出了利用手术切除, 患者衍生的病理 ft 进行三维表征和体外培养。新鲜的 ft 被转移到研究实验室并进…
Discussion
考虑到相关组织微环境对可靠的功能细胞和分子力学结果的重要性, 有必要找到适当的实验模型, 提供这种组织环境。本文介绍了纤维蛋白嵌入 ft 的体外pdr 培养模型, 可以研究 pdr 病理生理学在 pdr 临床样本的本机、复杂和多细胞环境中的机制。
协议中的关键步骤是适当的纤维蛋白凝胶形成、ft 的定位和染色过程中的适当清洗。由于纤维蛋白凝胶的形成主要取决于凝血?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者非常感谢赫尔辛基大学医院眼科糖尿病科和玻璃体视网膜外科的医疗和外科医生、护士和全体工作人员积极参与招聘工作。的病人。我们感谢生物素分子成像设备的成像设施。我们感谢阿纳斯塔西亚·切尔年科提供的出色技术援助。这项研究得到了芬兰科学院 (kl) 赠款的支持, 赫尔辛基大学 (kl)、sigrid juselius 基金会 (kl)、k. albin johansson 基金会 (kl)、芬兰癌症研究所 (kl)、karolinska 学院 (kl)、芬兰眼科基金会 (sl)、眼睛和组织银行基金会 (sl)、mary 和 georg c. ehrnrooth 基金会 (sl) 和 huch 临床研究赠款 (tyh2018127 (tyh2018127), TYH2018127, sl), 糖尿病研究基金会 (sl, kl, ak, eg) 以及生物医学博士方案 (eg)。
Materials
| Material | |||
| Microforceps | Medicon | 07.60.03 | Used for handling the FTs |
| Disposable Scalpels – Sterile | Swann-Morton | 0513 | Used for FT dissection |
| Culture dish, vented, 28 ml (60mm) | Greiner Bio-One | 391-3210 | Used for dissection and for testing fibrin gel formation |
| Cell culture plates, 12-well | Greiner Bio-One | 392-0049 | Used for FT dissection and whole-mount immunofluorescence |
| Reagent/centrifuge tube with screw cap, 15 mL | Greiner Bio-One | 391-3477 | |
| Reagent/centrifuge tube with screw cap, 50 mL | Greiner Bio-One | 525-0384 | |
| Millex-GV Syringe Filter Unit, 0.22 µm, PVDF | Millipore | SLGV033RS | Used to sterile-filter the fibrinogen solution |
| Syringe, 10 mL | Braun | 4606108V | Used to sterile-filter the fibrinogen solution |
| Polypropylene Microcentrifuge Tubes, 1.5 mL | Fisher | FB74031 | |
| Cell-Culture Treated Multidishes, 24-well | Nunc | 142475 | Used for casting the FT/fibrin gels for native FT characterization and ex vivo culture |
| Cell culture plates, 96-well, U-bottom | Greiner Bio-One | 392-0019 | Used for whole-mount immunofluorescence |
| Round/Flat Spatulas, Stainless Steel | VWR | 82027-528 | Used for whole-mount immunofluorescence |
| Coverslips 22x22mm #1 | Menzel/Fisher | 15727582 | Used for mounting |
| Microscope slides | Fisher | Kindler K102 | Used for mounting |
| Absorbent paper | VWR | 115-0202 | Used for mounting |
| Name | Company | Catalog Number | Comments |
| Reagents | |||
| PBS tablets | Medicago | 09-9400-100 | Used for preparing 1x PBS |
| Fibrinogen, Plasminogen-Depleted, Human Plasma | Calbiochem | 341578 | |
| Hanks Balanced Salt Solution | Sigma-Aldrich | H9394-500ML | Used for preparing the fibrinogen and TA solution |
| Fetal bovine serum | Gibco | 10270106 | Used for preparing the blocking solution |
| Human Serum | Sigma-Aldrich | H4522 | Aliquoted in -20 °C, thaw before preparing the ex vivo culture media |
| Gentamicin Sulfate 10mg/ml | Biowest | L0011-100 | |
| Endothelial cell media MV Kit | Promocell | C-22120 | Contains 500 ml of Endothelial Cell Growth Medium MV, 25 mL of fetal calf serum, 2 mL of endothelial cell growth supplement, 500 μL of recombinant human epidermal growth factor (10 μg/ mL) and 500 μL of hydrocortisone (1 g/ mL) |
| Sodium azide | Sigma-Aldrich | S2002 | Used for storage of the native and ex vivo cultured FTs. TOXIC: wear protective gloves and/or clothing, and eye and/or face protection. Use in fume hood. |
| Acetone | Sigma-Aldrich | 32201-2.5L-M | Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood. |
| Methanol | Sigma-Aldrich | 32213 | Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood. |
| Triton X-100 (octyl phenol ethoxylate) | Sigma-Aldrich | T9284 | Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing. |
| Hoechst 33342, 20mM | Life Technologies | 62249 | For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection. |
| VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000 | Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
| VECTASHIELD Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200 | Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
| Eukitt Quick-hardening mounting medium | Sigma-Aldrich | 03989-100ml | TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood. |
| Thrombin from bovine plasma, lyophilized powder | Sigma-Aldrich | T9549-500UN | Dissolve at 100 units/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw |
| Aprotinin from bovine lung, lyophilized powder | Sigma | A3428 | Dissolve at 50 mg/ mL, aliquote and store at -20 °C, avoid repeated freeze/ thaw |
| Name | Company | Catalog Number | Comments |
| Growth factors | |||
| Recombinant human VEGFA | R&D Systems | 293-VE-010 | 50 ng/ mL final concentration |
| Recombinant human VEGFC | R&D Systems | 752-VC-025 | 200 ng/ mL final concentration |
| Recombinant human TGFβ | Millipore | GF346 | 1 ng/ mL final concentration |
| Recombinant human bFGF | Millipore | 01-106 | 50 ng/ mL final concentration |
| Name | Company | Catalog Number | Comments |
| Primary antibodies | |||
| CD31 (JC70A) | Dako | M0823 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
| CD34 (QBEND10) | Dako | M716501-2 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
| CD45 (2B11+PD7/26) | Dako | M070129-2 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
| CD68 | ImmunoWay | RLM3161 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
| Cleaved caspase-3 (5A1E) | Cell Signalling | 9664 | Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
| ERG (EP111) | Dako | M731429-2 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
| GFAP | Dako | Z0334 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
| Ki67 | Leica Microsystems | NCL-Ki67p | Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
| Lyve1 | R&D Systems | AF2089 | Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab |
| NG2 | Millipore | AB5320 | Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab |
| Prox1 | ReliaTech | 102-PA32 | Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab |
| Prox1 | R&D Systems | AF2727 | Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab |
| VEGFR3 (9D9F9) | Millipore | MAB3757 | Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab |
| α-SMA (1A4) | Sigma | C6198 | Used at 1:400 dilution, Cy3 conjugated |
| Name | Company | Catalog Number | Comments |
| Secondary antibodies | |||
| Alexa Fluor488 Donkey Anti-Mouse IgG | Life Technologies | A-21202 | Used at 1:500 dilution |
| Alexa Fluor594 Goat Anti-Rabbit IgG | Invitrogen | A-11012 | Used at 1:500 dilution |
| Alexa Fluor568 Donkey anti-Goat IgG | Thermo Scientific | A-11057 | Used at 1:500 dilution |
| Alexa Fluor568 Goat anti-Rabbit IgG | Thermo Scientific | A-11036 | Used at 1:500 dilution |
| Alexa Fluor594 Chicken Anti-Goat IgG | Molecular Probes | A-21468 | Used at 1:500 dilution |
| Name | Company | Catalog Number | Comments |
| Microscopes | |||
| Axiovert 200 inverted epifluorescence microscope | Zeiss | For imaging of the fresh and fibrin-embedded FT | |
| SZX9 upright dissection stereomicroscope | Olympus | For FT dissection | |
| LSM 780 confocal microscope | Zeiss | For imaging of whole-mount immunostained FT | |
| AxioImager.Z1 upright epifluorescence microscope with Apotome | Zeiss | For imaging of whole-mount immunostained FT |
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Citazione di questo articolo
Gucciardo, E., Loukovaara, S., Korhonen, A., Lehti, K. An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy. J. Vis. Exp. (143), e59090, doi:10.3791/59090 (2019).