以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合
Summary
本手稿描述了如何在培养的上皮细胞单分子膜切口样病变, 方便模型伤口愈合在体外, 允许成像的共焦或激光扫描显微镜, 并能提供高质量定量和定性数据, 用于研究细胞行为和参与迁移的机制。
Abstract
细胞迁移是伤口愈合的一个必需的方面。在研究动物模型上制造人工伤口往往导致昂贵和复杂的实验程序, 但可能缺乏精确性。体外上皮细胞系的培养为研究创面愈合过程中细胞迁移行为和治疗对这些细胞的影响提供了一个合适的平台。上皮细胞的生理学在 non-confluent 条件下经常被研究;然而, 这种方法可能不像自然伤口愈合的条件。通过机械手段破坏上皮细胞的完整性产生了一个现实的模型, 但可能会阻碍分子技术的应用。因此, 显微技术是最理想的研究上皮细胞迁移的体外.在这里, 我们详细介绍了两种具体的方法, 人工伤刮法和人工迁移前分析法, 可以分别获得定量和定性的数据, 对上皮细胞的迁移性能。
Introduction
细胞迁移是需要的伤口愈合, 因为它是负责最后关闭的上皮间隙和恢复破坏表面1。在动物模型中执行人工伤口允许在近生理条件下复制这个复杂的过程2。然而, 这种方法往往导致昂贵和复杂的实验程序, 可能缺乏精确的研究不同的过程, 由于复杂的性质, 伤口愈合过程。
体外上皮细胞系的培养为研究这些细胞在创伤愈合中所起的作用以及治疗对细胞迁移行为的影响提供了一种有益的替代动物模型。利用 non-confluent 培养的分子技术, 经常研究上皮细胞的生理特性3,4,5,6;然而, 上皮完整性的破坏通常是通过细微的机械切口来实现的。在细胞培养中, 这意味着可忽略的细胞数可能暴露在伤口间隙, 它们代表了一个太小的样本分子生物学技术。然而, 这些病变可以在显微镜下进行研究, 利用一些上皮细胞系的固有迁移特性, 如水貂肺上皮细胞 (Mv1Lu) 或自发永生化的人角质形成细胞 (HaCaT)。线.
在这里, 我们描述了一种显微镜方法, 它是适合获得定量数据的上皮细胞迁移在伤口愈合的情况下3,4,7,8。此外, 我们提出的其他方法, 有助于研究的定性分子和形态学的变化发生在上皮膜的迁移。总的来说, 这些方法提供了一个框架来研究的动力学和形态学的变化涉及上皮细胞的行为和反应的治疗过程中伤口愈合。
Protocol
Representative Results
Discussion
在皮肤或粘膜的破坏, 屏障功能恢复的行动, 许多细胞类型, 包括成纤维细胞或上皮和免疫细胞。共同, 这些细胞经历了一个复杂的过程, 涉及细胞凋亡, 增殖, 分化, 重要的是, 成纤维细胞和上皮组织迁移, 这是最终的机制, 负责修复被破坏的组织和浅上皮间隙闭合1,12因此, 研究细胞迁移有助于精确描述上皮细胞的生理行为, 同时也确定伤口治疗的调节电位?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢实验室的老成员, 帮助改善和完善这些技术的实际状态: Dr. 西莉亚。Dr. 安娜 Mrowiec, Dr., 拉肯纳和 Dr.-加西亚。我们感谢医院 Clínico 体育馆圣女 Arrixaca 大力支持这些技术的发展。还有干杯卡洛斯三世, 基金 de 门多萨市 Sanitarias。计划国家 i + D + i 和干杯研究所-Subdirección 总 de Evaluación y 调值研究 (批准号: PI13/00794);www.isciii.es. Fondos 菲德 “manera de hacer 欧罗巴”。我们还感谢穆尔西亚、IMIB-Arrixaca 和 FFIS 的行政支助和援助。最后, 我们要特别感谢 Dr. 伊莎贝尔-马丁内斯-Argudo 和 Facultad de Ciencias Ambientales Bioquímica, 校园 Tecnológico de Fábrica 武器, 卡斯蒂利亚 la 恰尔, 托莱多为他们的亲切的支持自愿割让生物医学和生物技术实验室, 使本论文的拍摄部分成为可能。
Materials
| Dulbecco’s Modified Eagle Medium (DMEM) | Biowest, Nuaillé, France | L0102-500 | Optional 10 % FBS supplement |
| Eagles’s Minimum Essential Medium (EMEM) | Lonza | BE12 -662F | Optional 10 % FBS supplement |
| L-Glutamine | Lonza | BE17-605E | Use at 2 mM |
| Fetal Bovine Serum (FBS) | Thermo Fisher Scientific, Waltham, MA USA | DE17603A | |
| Trypsin-EDTA | Sigma-Aldrich, St Louis, MO, USA | T4049 | Dilute as appropriate |
| Poly-L-Lysine | Sigma-Aldrich, St Louis, MO, USA | P9155 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) (10x) | Gibco by Life Technologies | 14200-067 | Dilute to 1x |
| 24-well culture plates | BD FALCON//SARSTED | 734-0020 | |
| 6-well culture plates | SARSTEDT | 83-3920 | |
| Epidermal Growth Factor (EGF) | Sigma-Aldrich, St Louis, MO, USA | E9644 | Used 10 ng/mL |
| Round cover glass | MENZEL-GLÄSER | MENZCB00120RA020 | SHORT DEPTH OF FIELD |
| Reinforced razor blade no. 743 | Martor (through VWR) | MARO743.50 | |
| 200 µl sterile aerosol pipet tips | VWR | 732-0541 | |
| 20 µl sterile aerosol pipet tips | VWR | 732-0528 | |
| Digital camera coupled phase contrast microscope | Motic Spain | Moticam camera 2300 3.0 M Pixel USB 2.0; Motic Optic AE31 | |
| Confocal microscope | ZEISS Microimaging, Germany | LSM 510 META | |
| 10 cm Culture dish | BD FALCON | 353003 | |
| Rabbit polyclonal anti c-Jun antibody | Santa Cruz Biotechnology | sc-1694 | Used 1:100 |
| Anti-rabbit IgG (polyclonal goat ) AF 488 | Invitrogen | A11008 | Used 1:400 |
| Hoechst-33258 | Sigma-Aldrich, St Louis, MO, USA | 14530 | Used 1:1000 |
| Alexa Fluor 594 phalloidin (in methanol) (red) | Invitrogen | A12381 | Used 1:100 |
| Bovine Serum Albumin | Santa Cruz Biotechnology | SC-2323 | |
| Triton X-100 | Sigma-Aldrich, St Louis, MO, USA | T9284 | |
| Skim milk | BD DIFCO | 232100 | |
| ImageJ | National Institutes of Health, USA | Release 1.50i | |
| Zen LSM 510 image processing software | ZEISS Microimaging, Germany | Release 5.0 SP 1.1 |
References
- Singer, A. J., Clark, R. A. Cutaneous wound healing. N Engl J Med. 341 (10), 738-746 (1999).
- Davidson, J. M. Animal models for wound repair. Arch Dermatol Res. 290, S1-S11 (1998).
- Alcaraz, A., et al. Amniotic Membrane Modifies the Genetic Program Induced by TGFss, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds. PLoS One. 10 (8), e0135324 (2015).
- Ruiz-Canada, C., et al. Amniotic membrane stimulates cell migration by modulating Transforming Growth Factor-beta signaling. J Tissue Eng Regen Med. , (2017).
- Schmierer, B., Hill, C. S. TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol. 8 (12), 970-982 (2007).
- Hu, Y. L., et al. FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells. Sci Rep. 4, 6024 (2014).
- Martinez-Mora, C., et al. Fibroin and sericin from Bombyx mori silk stimulate cell migration through upregulation and phosphorylation of c-Jun. PloS one. 7 (7), e42271 (2012).
- Bernabe-Garcia, A., et al. Oleanolic acid induces migration in Mv1Lu and MDA-MB-231 epithelial cells involving EGF receptor and MAP kinases activation. PLoS One. 12 (2), e0172574 (2017).
- Huang, H. L., et al. Trypsin-induced proteome alteration during cell subculture in mammalian cells. J Biomed Sci. 17, 36 (2010).
- Liberio, M. S., Sadowski, M. C., Soekmadji, C., Davis, R. A., Nelson, C. C. Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior. PLoS One. 9 (11), e112122 (2014).
- Pierreux, C. E., Nicolas, F. J., Hill, C. S. Transforming growth factor beta-independent shuttling of Smad4 between the cytoplasm and nucleus. Mol Cell Biol. 20 (23), 9041-9054 (2000).
- Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., Tomic-Canic, M. Growth factors and cytokines in wound healing. Wound repair and regeneration: official publication of the Wound Healing Society [and] the European Tissue Repair Society. 16 (5), 585-601 (2008).
- Ansell, D. M., Holden, K. A., Hardman, M. J. Animal models of wound repair: Are they cutting it?. Exp Dermatol. 21 (8), 581-585 (2012).
- Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. Wound repair and regeneration. Nature. 453 (7193), 314-321 (2008).
- Insausti, C. L., et al. Amniotic membrane induces epithelialization in massive posttraumatic wounds. Wound Repair Regen. 18 (4), 368-377 (2010).
- Wu, F., et al. Cell cycle arrest in G0/G1 phase by contact inhibition and TGF-beta 1 in mink Mv1Lu lung epithelial cells. Am J Physiol. 270 (5 Pt 1), L879-L888 (1996).
- Boukamp, P., et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 106 (3), 761-771 (1988).
- Marshall, J., Wells, C. M., Parsons, M. . Cell Migration: Developmental Methods and Protocols. , 97-110 (2011).
- Nyegaard, S., Christensen, B., Rasmussen, J. T. An optimized method for accurate quantification of cell migration using human small intestine cells. Metabolic Engineering Communications. 3, 76-83 (2016).
- Szybalski, W., Iyer, V. N. Crosslinking of DNA by Enzymatically or Chemically Activated Mitomycins and Porfiromycins, Bifunctionally “Alkylating” Antibiotics. Fed Proc. 23, 946-957 (1964).
- Tomasz, M. Mitomycin C: small, fast and deadly (but very selective). Chem Biol. 2 (9), 575-579 (1995).
- Sherry, D. M., Parks, E. E., Bullen, E. C., Updike, D. L., Howard, E. W. A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption. Cell Adh Migr. 7 (6), 469-475 (2013).
- Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J. 5 (7), 2068-2077 (1991).
- Lampugnani, M. G. Cell migration into a wounded area in vitro. Methods Mol Biol. 96, 177-182 (1999).
