从羊膜中提取Lumican及其储存温度的测定
Summary
本协议描述了从羊膜（AM）中提取lumican及其储存条件作为AM提取物（AME）在-20°C，4°C和室温（RT）下6，12，20和32天以量化其蛋白质和lumican浓度。
Abstract
Lumican是人羊膜（AM）中一种富含亮氨酸的小蛋白聚糖，可促进角膜上皮化和胶原纤维的组织，保持角膜透明。本工作提出了一种从AM中提取蛋白质得到Lumican的方法。此外，还评估了在不同温度和时间段下储存的AM提取物（AME）中Lumican的稳定性。解冻100mgAM并机械去上皮化。将去上皮化的AM冷冻并粉碎，直至获得细粉，用2.5mL盐水缓冲液与蛋白酶抑制剂溶解并离心进行蛋白质提取。收集上清液并在-20°C、4°C和室温（RT）下保存6、12、20和32天。之后，在每个AME中量化Lumican。该技术允许从AM中提取lumican的可访问和可获取的方案。Lumican浓度受储存时间和温度条件的影响。Lumican在AME中在-20°C和4°C下储存12天明显高于其他AME。这种lumican提取可用于开发治疗方法和药物解决方案。需要进一步的研究来确定AME发光胶在再上皮化和伤口愈合过程中的用途。
Introduction
角膜影响最常用的治疗方法之一是羊膜移植;然而，近年来，出现了使用羊膜组织的各种成分作为替代和辅助治疗的新建议。在研究最多的AM成分中，是从AM提取物（AME）中获得的成分1，2，3，4，5，6，7。AM含有多种可溶性因子，例如抗血管生成蛋白，白细胞介素（IL），金属蛋白酶（TIMP）的组织抑制剂，由TSG-6介导的抑制中性粒细胞细胞外陷阱的抗炎蛋白，生长因子：表皮生长因子（EGF），转化生长因子（TGF）（α和β），角质形成细胞生长因子（KGF），肝细胞生长因子（HGF）和Lumican，通过调节胶原纤维生成来维持角膜透明度1，2，3，4，5，6，7，8，9.
Lumican是一种富含亮氨酸的小蛋白聚糖（SLRP），是角膜基质基质中间质胶原酶的主要细胞外成分之一，负责组织胶原纤维并保持角膜透明度4，10，11。蛋白聚糖是细胞外基质（ECM）中的分子，是进行细胞信号传导和维持细胞内稳态的主要分子12。据报道，ECM蛋白在伤口愈合过程中驱动细胞增殖，分化和迁移过程11。
证据表明Lumican可能参与角膜再上皮化过程。Saika 等人在一项研究中表明，角膜损伤后，可以在损伤后的前 8 小时至最多 3 天内在角膜角质细胞中检测到 lumican。在第二天和第三天呈现最高浓度的lumican，该蛋白聚糖随后在第七天无法检测到13。这些数据表明Lumican参与角膜再上皮化过程的激活。另一方面，在另一项研究中，据报道，没有发光胶会延迟再上皮化;有趣的是，添加Lumican可以加速再上皮化过程4，11，13。同样，最近的一项研究报告说，Lumican可以调节角膜缘成纤维细胞14的炎症功能，这表明Lumican作为炎症，抗纤维化和再上皮化反应的调节剂发挥作用。同样，lumican可以通过与Fas-FasL等信号分子相互作用来调节角膜反应。此外，在敲除Lum–/-小鼠模型中没有Lumican表明缺乏lumican信号传导会阻止充分的角膜修复15。
该方法主要旨在展示一种从AM中提取lumican的可行且平易近人的方法。与以前的研究相比，使用这种有利的lumican提取方法可以获得相似浓度的蛋白质，从而减少了处理时间，并使研究人员更方便16。此外，这种AME发光胶可用作角膜修复和再上皮化过程的佐剂。
Protocol
所有实验程序均已获得机构审查委员会的批准（项目编号：CEI-2020/06/04）。AM是从瓦伦西亚孔德羊膜库（来自去识别的人类受试者）获得的，该数据库是按照Chávez-García等人的描述制备的17。 1.羊膜提取物的制备 从羊膜库获取 100 毫克 AM。注意：根据之前的报告，50 毫克 AM 总共分泌 10 ng/mL 的卢米康14。为了获得更高浓?…
Representative Results
结果报告为标准偏差 （SD） ±平均值。进行学生的 t检验和方差分析（方差分析）。 < 0.05 的 P 值被认为具有统计学意义。使用统计软件进行统计分析（见 材料表）。 AME中的总蛋白质量受时间和储存条件的影响。所有AME的基础蛋白浓度相似;总蛋白的范围为2.7±0.3μg/mL，评估的样品之间没有显着差异。然而，当样品储存12、20和32天时，观察到蛋白质浓?…
Discussion
本研究分析了AME中Lumican的存在及其在不同储存条件下的稳定性的直接相关性。有趣的是，当AME中的总蛋白质浓度被量化时，储存后蛋白质浓度增加。有证据表明，有三种机制可以改变冷冻储存中的蛋白质浓度：冷变性、溶质的冷冻浓度和冰诱导的蛋白质结构部分展开19。由于样品中液相的结晶，冷冻过程可能会影响储存样品上的蛋白质浓度。结果表明，这可能是冷冻浓度的过程?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者没有相互竞争的经济利益。
Materials
| 1 N H2SO4 stop solution | R&D Systems | DY994 | |
| 100 μL micropipette | Eppendorf | ||
| 1000 μL micropipette | Eppendorf | ||
| 15 mm Petri dish | Symlaboratorios | ||
| 18 G Needle (1.2 mm x 40 mm) | BD Becton Dickinson | 305211 | |
| 2 mL microcentrifuge tube | Eppendorf | Z606340 | |
| 20 mL plastic syringe | BD Becton Dickinson | 302562 | |
| 20 μL micropipette | Eppendorf | ||
| 20-200 μL micropipette | Eppendorf | ||
| 5 mL microcentrifuge tube | Eppendorf | 30119401 | |
| 96-well microplate | SARSTEDT | 821581 | |
| Aluminum foil | N/A | N/A | |
| Amniotic membrane | Instituto de Oftalmologia Conde de Valenciana Amnion Bank | 100 mg | |
| Balanced salt solution | Bausch + Lomb | BSS-403802 | |
| Beaker | N/A | N/A | |
| BioRender | BioRender | figures design | |
| Compact Rocker | BioRad | 970822DD | Mod. 5202SD-BIO |
| complete, EDTA-free, Protease inhibitor cocktail tablets |
Roche | 11 873 580 001 | Protease Inhibitor |
| Daiggner vortex Genie 2 | A.Daigger & Co. , INC | 22220A | |
| Dispase II | Gibco | 17105-041 | |
| ELISA plate spectrometer | Thermo Labsystems | 35401106 | Multiscan |
| Freezer | |||
| GraphPad Prism | GraphPad Software, Inc | version 9 | statistical analysis and graphic program |
| Human lumican DuoSet ELISA kit | R&D Systems | DY2846-05 | includes human Lumican capture antibody |
| Incubator | Forma Scientific | 3326 S/N 36481-7002 | |
| Inverted light Microscope | Olympus | 6A13921 | to confirm de-epithelialization Mod.CK2 |
| Laminar flow hood | Forma Scientific | 14753-567 | Mod.1184 |
| Liquid nitrogen | N/A | N/A | |
| Mortar | N/A | N/A | |
| Multi-channel pipettor | Eppendorf | ||
| Nitrogen Tank | Thermo Scientific | Mod. Biocan 20 | |
| Paper towels | N/A | N/A | |
| Phosphate-buffered saline | R&D Systems | DY006 | |
| Pierce Modified Lowry Protein Assay Kit | Thermo Scientific | 23240 | |
| Plate sealers | R&D Systems | DY992 | |
| Reagent diluent | R&D Systems | DY995 | 1% BSA in PBS, pH 7.2-7.4, 0.2 μm filtered |
| Refrigerated centrifuge | centurion scientific Ltd | 15877 | Mod. K2015R |
| Rubber policeman cell scraper | NEST | 710001 | for mechanical de-epithelialization |
| Scalpel knife | Braun | BB521 | No. 10 or 21 |
| Streptavidin-HRP 40-fold concentrated | R&D Systems | part 893975 | |
| Substrate tetramethylbenzidine (TMB) solution | R&D Systems | DY999 | |
| Toothed tweezers | Invent Germany | 6b | inox |
| Ultrapure water | PISA | ||
| Wash buffer | R&D Systems | WA126 | 0.05% Tween 20 in PBS, pH 7.2-7.4 |
References
- Jirsova, K., Jones, G. Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review. Cell and Tissue Banking. 18 (2), 193-204 (2017).
- Witherel, C., Yu, T., Concannon, M., Dampier, W., Spiller, K. Immunomodulatory effects of human cryopreserved viable amniotic membrane in a pro-inflammatory environment in vitro. Cellular and Molecular Bioengineering. 10 (5), 451-462 (2017).
- Ruiz-Cañada, C., et al. Amniotic membrane stimulates cell migration by modulating transforming growth factor-β signalling. Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 808-820 (2017).
- Yeh, L., et al. Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing. Investigative Opthalmology & Visual Science. 46 (2), 479 (2005).
- Navas, A., et al. Anti-Inflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and their potential in corneal repair. Stem Cells Translational Medicine. 7 (12), 906-917 (2018).
- Magaña-Guerrero, F., Domínguez-López, A., Martínez-Aboytes, P., Buentello-Volante, B., Garfias, Y. Human amniotic membrane mesenchymal stem cells inhibit neutrophil extracellular traps through TSG-6. Scientific Reports. 7, 12426 (2017).
- Garfias, Y., Zaga-Clavellina, V., Vadillo-Ortega, F., Osorio, M., Jimenez-Martinez, M. Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells. Immunological Investigations. 40 (2), 183-196 (2010).
- Koob, T., et al. Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing. International Wound Journal. 10 (5), 493-500 (2013).
- Miyagi, H., Thomasy, S., Russell, P., Murphy, C. The role of hepatocyte growth factor in corneal wound healing. Experimental Eye Research. 166, 49-55 (2018).
- Chen, S., Mienaltowski, M., Birk, D. Regulation of corneal stroma extracellular matrix assembly. Experimental Eye Research. 133, 69-80 (2015).
- Karamanou, K., Perrot, G., Maquart, F., Brézillon, S. Lumican as a multivalent effector in wound healing. Advanced Drug Delivery Reviews. 129, 344-351 (2018).
- Theocharis, A., et al. Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer. FEBS Journal. 281 (22), 5023-5042 (2014).
- Saika, S., et al. Role of lumican in the corneal epithelium during wound healing. Journal of Biological Chemistry. 275 (4), 2607-2612 (2000).
- Domínguez-López, A., et al. Amniotic membrane conditioned medium (AMCM) reduces inflammatory response on human limbal myofibroblast, and the potential role of lumican. Molecular Vision. 27, 370-383 (2021).
- Vij, N., Roberts, L., Joyce, S., Chakravarti, S. Lumican regulates corneal inflammatory responses by modulating Fas-Fas Ligand signaling. Investigative Opthalmology & Visual Science. 46 (1), 88 (2005).
- Mahbod, M., et al. Amniotic membrane extract preparation: What is the best method. Journal of Ophthalmic and Vision Research. 9 (3), 314-319 (2014).
- Chávez-García, C., et al. Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience. Cell and Tissue Banking. 17 (2), 261-268 (2015).
- Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase. Journal of Investigative Dermatology. 93 (2), 287-290 (1989).
- Bhatnagar, B. S., Bogner, R. H., Pikal, M. J. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharmaceutical Development and Technology. 12 (5), 505-523 (2007).
- McClain, A. K., McCarrel, T. M. The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. BMC Veterinary Research. 15 (1), 292 (2019).
- Tamhane, A., et al. Evaluation of amniotic membrane transplantation as an adjunct to medical therapy as compared with medical therapy alone in acute ocular burns. Ophthalmology. 112 (11), 1963-1969 (2005).
- Shtein, R., et al. Autologous serum-based eye drops for treatment of ocular surface disease. Ophthalmology. 127 (1), 128-133 (2020).
- Shahriari, H., Tokhmehchi, F., Reza, M., Hashemi, N. Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model. Cornea. 27 (10), 1148-1150 (2008).
- Schuerch, K., Baeriswyl, A., Frueh, B., Tappeiner, C. Efficacy of amniotic membrane transplantation for the treatment of corneal ulcers. Cornea. 39 (4), 479-483 (2019).
- Chen, H., et al. Amniotic membrane transplantation for persistent corneal ulcers and perforations in acute fungal keratitis. Cornea. 25 (5), 564-572 (2006).
- Guo, Q., et al. A comparison of the effectiveness between amniotic membrane homogenate and transplanted amniotic membrane in healing corneal damage in a rabbit model. Acta Ophthalmologica. 89 (4), 315-319 (2011).
- Sabater, A., Perez, V. Amniotic membrane use for management of corneal limbal stem cell deficiency. Current Opinion in Ophthalmology. 28 (4), 363-369 (2017).
- Ahmad, T., et al. Autolysis of bovine skin, its endogenous proteases, protease inhibitors and their effects on quality characteristics of extracted gelatin. Food Chemistry. 265, 1-8 (2018).
- Mullegama, S. V., et al. Nucleic acid extraction from human biological samples. Methods in Molecular Biology. 1897, 359-383 (2019).
- Skog, M., et al. The effect of enzymatic digestion on cultured epithelial autografts. Cell Transplantation. 28 (5), 638-644 (2019).
Cite This Article
Haro-Morlett, L., Magaña-Guerrero, F. S., Volante, B. B., Garfias, Y. Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature. J. Vis. Exp. (188), e64460, doi:10.3791/64460 (2022).