创面愈合研究的前活体角膜器官培养模型
Summary
介绍了一种适用于伤口愈合研究的体外角膜器官培养模型的方案。该模型系统可用于评估药物在有组织的3d 多细胞环境中促进再生愈合或药物毒性的效果。
Abstract
角膜已被广泛用作研究伤口愈合的模型系统。在二维 (2d) 和三维培养中产生和利用原代哺乳动物细胞的能力, 不仅产生了大量关于角膜生物学的信息, 而且还产生了大量关于伤口愈合、肌成纤维细胞生物学和一般疤痕的信息。.该方案的目标是一个检测系统, 用于定量肌成纤维细胞的发展, 其特点是疤痕。我们展示了一个角膜器官培养的体外模型使用猪眼。在这个前角膜切除术伤口, 角膜仍然在地球上受伤的圆形刀片称为树状。大约 1, 1 的角膜前部的塞子被切除, 包括上皮, 基底膜, 和前部的间质。受伤后, 角膜从地球上被切割, 安装在胶原蛋白基地, 并在补充血清游离培养基中培养两周, 并具有稳定的维生素 c, 以增加细胞增殖和细胞外基质分泌的居住性成纤维细胞。肌成纤维细胞在角膜愈合中的激活是明显的。该模型可用于检测创面闭合、肌成纤维细胞和纤维化标志物的发育以及毒理学研究。此外, 小分子抑制剂以及脂介导的 sirna 转染对基因敲除的影响可以在该系统中进行测试。
Introduction
由于受伤、外伤或感染而导致的角膜损伤会导致虚弱的不平衡和永久性视力下降。因此, 迫切需要确定可针对治疗干预的途径。目前的治疗选择有限, 主要包括角膜移植, 世界各地的病人都无法获得角膜移植。人 (图 1) 和动物角膜都可用于2d 和3d 细胞培养研究1,2。不适合移植的人体尸体角膜可以从眼库或中央组织库 (国家疾病研究相互交流 (ndri)) 获得, 动物眼睛可以从屠宰场获得。原代角膜上皮细胞、基质成纤维细胞以及最近的内皮细胞, 可以从这些组织中分离和培养, 用于伤口愈合和毒理学研究 3,4,5。除了了解致盲眼病的分子基础、组织的可获得性和培养原代细胞的能力外, 角膜也是重要的研究模型系统。角膜是理想的测试剂对疤痕的影响, 因为正常角膜是透明的, 某些类型的伤口造成不透明或纤维化疤痕 (回顾在 6)。几种体内角膜伤口愈合模型也被广泛用于疤痕研究1。利用较少的是体外角膜伤口愈合模型7,8 , 这里详细描述。该方法的目的是量化在三维多细胞角膜外模型系统中由纤维化的制造商所特有的疤痕结局。
角膜上皮伤, 不违反上皮基底膜通常关闭在 24-72 h9。受伤后不久, 上皮边缘的细胞开始扩散并迁移到上皮自由表面, 以重建上皮屏障功能。这一活动随后依次激活角膜基底细胞增殖, 并在稍后阶段, 前驱细胞位于外缘膜区, 以实现上皮细胞质量的恢复 10,11.这些伤口往往愈合而没有疤痕。然而, 穿透基底膜进入间质的伤口往往会导致疤痕形成1。角膜间质损伤后, 间质由多个来源的细胞组成, 包括分化的常驻间质细胞以及骨髓源性纤维细胞 12,13,14。纤维化疤痕的特点是肌成纤维细胞在愈合伤口的持久性。这些病理性肌成纤维细胞表现出增加的粘附通过整合整合在局灶性粘连, 收缩α-平滑肌肌动蛋白 (α-sma) 应力纤维, 并局部激活细胞外基质 (ecm) 隔离后期转化的生长因子-β (tgfβ)。上皮细胞的分化, 称为上皮间充质转变 (emt), 也可能有助于瘢痕的形成 6.
细胞分化和损伤后细胞凋亡之间存在微妙的平衡。由于基底膜的断裂, 生长因子, 如血小板衍生生长因子 (pdgf) 和 tgfβ的生长因子, 如血小板衍生生长因子 (pdgf) 和 tgfβ, 由于眼泪和上皮沐浴的间质, 诱导肌成纤维细胞分化, 一个持续的自体循环 tgfβ, 和分泌不组织的纤维化 ecm15,16。肌成纤维细胞在愈合的伤口中的持续存在会促进角膜的雾霾和疤痕 (图 2)。然而, 在再生愈合的伤口, 虽然肌成纤维细胞的发展, 他们凋亡, 因此是缺席或明显减少的数量在愈合的组织 (回顾在参考6,10)。因此, 对纤维化疤痕的研究至少在一定程度上集中在靶向分子上, 以防止肌成纤维细胞过度发育或肌成纤维细胞持久性17,18。由于肌成纤维细胞的持久性在所有组织19中都有疤痕和纤维化疾病的特点, 角膜可能是研究纤维化的一般细胞机制的模型系统。
在我们的模型系统中, 角膜在地球上的时候被一个称为树状的圆柱形刀片击伤。人和猪角膜可以用6或7毫米的头孢菌伤;对于兔角膜是首选6毫米树状。猪角膜的大小与人角膜相似。因为猪角膜具有成本效益, 而且数量广泛, 因此通常用于器官培养。此外, 与人发生反应的抗体和 sirna 一直与猪7交叉反应。伤人后, 角膜被切割出地球与石灰完整, 并安装在琼脂胶原蛋白基地。角膜在无血清介质中培养, 并加入稳定的维生素 c, 以模拟成纤维细胞增殖和 ecm 沉积20。诱导肌成纤维细胞形成均不需要血清的添加和生长因子.在经过两周的培养后, 角膜定期固定和处理组织学。对于基因敲除, 或测试药物对伤口愈合的影响, 伤口可在伤后用 sirna 治疗, 伤后7人, 或可在培养基中添加可溶性剂, 分别为 8。
Protocol
1. 器官文化 准备 准备琼脂解决方案, 如下所示。在一个小烧瓶中, 在 dmem-f12 中制备1% 琼脂和 1 mgml 牛胶原蛋白, 最高可达20毫升。在热盘上煮沸。将溶液放入50毫升的锥形管中。将管子放在热水澡中的热板上, 以防止溶液凝固。 根据材料表中提供的成分准备补充的无血清介质 (ssfm)。请注意:要准备的 ssfm 的必要数量取决于要处理的角?…
Representative Results
免疫组织化学是分析体外创面愈合实验成功的主要方法。图 4显示对照组织中的上皮和前间质 (图 4a, 4A)。受伤6小时后, 上皮无 (图 4c, 4C)。如预期的伤势后六天, 上皮已重新生长 (图 4e, 4E)。该组织对α-平滑肌肌动蛋白 (α-sma) 进行免疫抑制, 其表达…
Discussion
该协议描述了一个模型, 用于研究在自然分层三维环境中的伤口愈合。使用器官培养作为细胞培养和体内研究之间的中间体, 大大降低了成本, 也减少了活体动物的程序。其他3d 模型对该领域有很大的好处, 包括由原代人类角膜成纤维细胞 2或这些相同的细胞嵌入在由动物衍生胶原蛋白31制成的凝胶中的自合成胶原蛋白凝胶.器官培养模型系统对于测试假定的治疗剂…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 nih-nei r01 ey024942、预防失明研究、州立医科大学无限制研究基金和狮子区20-y 的支持, 在显微镜核心进行了石蜡切片的显微镜和图像分析。组织学幻灯片的准备工作是在西奈山伊坎医学院的生物存储和病理核心进行的。
Materials
| PBS | Gibco | 10-010-023 | |
| Pen Strep | MP Biomedicals | 91670049 | |
| Bovine Collagen Solution | Advance Biomatrix | 5005 | |
| Pig eyes with lids attached | Pel-freeze, Arkansas | N/A | |
| 6.0 mm trephine | Katena | K28014 | |
| Surgical Blade | Personna | 0.009 | |
| Small scissor | Fisher | 895110 | |
| Forceps | Fisher | 08953-F | |
| Kim Wipes | Kimberly-Clark™ 34120 | 06-666 | |
| 60 mm cell culture dishes | Falcon | 08-772B | |
| Supplemented Serum- Free media (SSFM) | Add all of the following components to DMEM/F-12: ITS, RPMI, Glutathione, L-Glutamine, MEM Non essential amino acids, MEM Sodium Pyruvate, ABAM, Gentamicin, Vitamin C. | ||
| DMEM/F-12 | Gibco | 11330 | |
| ITS Liquid Media Supplement | Sigma | I3146 | 100X |
| RPMI 1640 Vitamins Solution | Sigma | R7256 | 100X |
| Glutathione | Sigma | G6013 | Use at 1 µg/mL. Freeze aliquots; do not reuse after thawing. |
| 1% L-glutamine solution | Gibco | 25030-081 | 100X |
| MEM Non-essential amino acids solution | Gibco | 11140 | 100X |
| MEM Sodium pyruvate solution | Gibco | 11360 | 1 M Stocks (1000X) and freeze in single use aliquits. Use from freezer each time media is made. |
| ABAM | Sigma | A7292 | 100X |
| Gentamicin | Sigma | 30-005-CR | 200X |
| Vitamin C | Wako | 070-0483 | 2-0-aD Glucopyranosyl-Ascorbic Acid. 1 mM stocks (1000x) |
| 10% Iodine | Fisher Chemical | SI86-1 | |
| Tissue Path Cassettes | Fisher | 22-272416 | |
| Normal Goat Serum (NGS) | Jackson Immuno Research | 005-000-121 | We use 3% NGS |
| Mounting Media | Thermo Scientific | TA-030-FM | |
| Safe Clear | Fisher | 314-629 | |
| Ethyl Alcohol | Ultra Pure | 200CSGP | 200 Proof, diluted at 100%, 70%, 50%) |
| Sodium citrate | Fisher | BP327 | 10mM, pH 6.4 |
| Hematoxylin | EMD Millipore | M10742500 | |
| Bluing agent | Ricca Chemical Company | 220-106 | |
| 1% Triton X-100 | Fisher | 9002-93-1 | Diluted in PBS |
| 0.1% Tween 20 | Fisher | BP337 | Diluted in PBS |
| 3% Hydrogen Peroxide | Fisher | H324 | |
| DAB Kit | Vector Laboratories | SK-4100 | |
| Agar | Fisher | BP1423-500 | Agar solution: prepare 1% agar and 1 mg/mL bovine collagen in DMEM-F12 up to 20 mL |
| Parafilm | Bermis | 13-374-12 | |
| Moist Chamber | Use any chamber, cover it with wet Wipe Tissue and then put a layer of Parafilm over it. | ||
| Lipofectamine 2000 | |||
| Qiagen RNAprotect Cell Reagent | Qiagen | 76104 | |
| Ambion PureLink RNA Mini Kit | Thermo Scientific | 12183018A | |
| Anti-Fibronectin-EDA Antibody | Sigma | F6140 | 1:200 Diluted in 3% normal goat serum |
| Anti-alpha smooth muscle actin Antibody | Sigma | A2547 or C6198 (cy3 conjugated) | 1:200 Diluted in 3% normal goat serum |
| Permafluor | Thermo Scientific | TA-030-FM | |
| DAPI | Invitrogen | P36931 | |
| Gt anti -MS IgG (H+L) Secondary Antibody, HRP | Invitrogen | 62-6520 | 1:100 diluted in 3% normal goat serum (for a-SMA, DAB staining) |
| Gt anti -MS IgM (H+L) Secondary Antibody, HRP | Thermo Scientific | PA1-85999 | 1:100 diluted in 3% normal goat serum (for FN-EDA, DAB staining) |
| Gt anti -MS IgG (H+L) Secondary Antibody, Cy3 | Jackson Immuno Research | 115-165-146 | 1:200 Diluted in 3% normal goat serum (for a-SMA, Fluorescence staining) |
| Zeiss Axioplan2 | Zeiss | Microscope | |
| SPOT-2 | Diagnostic Instruments, Sterling Heights, Michigan | CCD camera | |
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Citazione di questo articolo
Castro, N., Gillespie, S. R., Bernstein, A. M. Ex Vivo Corneal Organ Culture Model for Wound Healing Studies. J. Vis. Exp. (144), e58562, doi:10.3791/58562 (2019).