Name: lumican extraction from amniotic membrane and determination of its storage temperature
Uploaded: 2022-10-14T14:30:00
Description: Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

作者没有相互竞争的经济利益。

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
<ol><li>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?...

<ol>
	<li>Jirsova, K., Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+in+ophthalmology:+properties,+preparation,+storage+and+indications+for+grafting-a+review.">Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.</a> Cell and Tissue Banking. 18 (2), 193-204 (2017).</li><li>Witherel, C., Yu, T., Concannon, M., Dampier, W., Spiller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+human+cryopreserved+viable+amniotic+membrane+in+a+pro-inflammatory+environment+in+vitro.">Immunomodulatory effects of human cryopreserved viable amniotic membrane in a pro-inflammatory environment in vitro.</a> Cellular and Molecular Bioengineering. 10 (5), 451-462 (2017).</li><li>Ruiz-Ca&#241;ada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+stimulates+cell+migration+by+modulating+transforming+growth+factor-&#946;+signalling.">Amniotic membrane stimulates cell migration by modulating transforming growth factor-&#946; signalling.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 808-820 (2017).</li><li>Yeh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+lumican+glycoprotein+purified+from+human+amniotic+membrane+promotes+corneal+epithelial+wound+healing.">Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.</a> Investigative Opthalmology &amp; Visual Science. 46 (2), 479 (2005).</li><li>Navas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Inflammatory+and+anti-fibrotic+effects+of+human+amniotic+membrane+mesenchymal+stem+cells+and+their+potential+in+corneal+repair.">Anti-Inflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and their potential in corneal repair.</a> Stem Cells Translational Medicine. 7 (12), 906-917 (2018).</li><li>Maga&#241;a-Guerrero, F., Dom&#237;nguez-L&#243;pez, A., Mart&#237;nez-Aboytes, P., Buentello-Volante, B., Garfias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amniotic+membrane+mesenchymal+stem+cells+inhibit+neutrophil+extracellular+traps+through+TSG-6.">Human amniotic membrane mesenchymal stem cells inhibit neutrophil extracellular traps through TSG-6.</a> Scientific Reports. 7, 12426 (2017).</li><li>Garfias, Y., Zaga-Clavellina, V., Vadillo-Ortega, F., Osorio, M., Jimenez-Martinez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+is+an+immunosuppressor+of+peripheral+blood+mononuclear+cells.">Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells.</a> Immunological Investigations. 40 (2), 183-196 (2010).</li><li>Koob, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+dehydrated+human+amnion/chorion+composite+graft:+implications+for+chronic+wound+healing.">Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.</a> International Wound Journal. 10 (5), 493-500 (2013).</li><li>Miyagi, H., Thomasy, S., Russell, P., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+hepatocyte+growth+factor+in+corneal+wound+healing.">The role of hepatocyte growth factor in corneal wound healing.</a> Experimental Eye Research. 166, 49-55 (2018).</li><li>Chen, S., Mienaltowski, M., Birk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+corneal+stroma+extracellular+matrix+assembly.">Regulation of corneal stroma extracellular matrix assembly.</a> Experimental Eye Research. 133, 69-80 (2015).</li><li>Karamanou, K., Perrot, G., Maquart, F., Br&#233;zillon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+as+a+multivalent+effector+in+wound+healing.">Lumican as a multivalent effector in wound healing.</a> Advanced Drug Delivery Reviews. 129, 344-351 (2018).</li><li>Theocharis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+interactions:+focus+on+proteoglycan-proteinase+interplay+and+pharmacological+targeting+in+cancer.">Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer.</a> FEBS Journal. 281 (22), 5023-5042 (2014).</li><li>Saika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lumican+in+the+corneal+epithelium+during+wound+healing.">Role of lumican in the corneal epithelium during wound healing.</a> Journal of Biological Chemistry. 275 (4), 2607-2612 (2000).</li><li>Dom&#237;nguez-L&#243;pez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+conditioned+medium+(AMCM)+reduces+inflammatory+response+on+human+limbal+myofibroblast,+and+the+potential+role+of+lumican.">Amniotic membrane conditioned medium (AMCM) reduces inflammatory response on human limbal myofibroblast, and the potential role of lumican.</a> Molecular Vision. 27, 370-383 (2021).</li><li>Vij, N., Roberts, L., Joyce, S., Chakravarti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+regulates+corneal+inflammatory+responses+by+modulating+Fas-Fas+Ligand+signaling.">Lumican regulates corneal inflammatory responses by modulating Fas-Fas Ligand signaling.</a> Investigative Opthalmology &amp; Visual Science. 46 (1), 88 (2005).</li><li>Mahbod, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+extract+preparation:+What+is+the+best+method.">Amniotic membrane extract preparation: What is the best method.</a> Journal of Ophthalmic and Vision Research. 9 (3), 314-319 (2014).</li><li>Ch&#225;vez-Garc&#237;a, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmic+indications+of+amniotic+membrane+transplantation+in+Mexico:+an+eight+years+Amniotic+Membrane+Bank+experience.">Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience.</a> Cell and Tissue Banking. 17 (2), 261-268 (2015).</li><li>Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispase,+a+neutral+protease+from+Bacillus+polymyxa,+is+a+powerful+fibronectinase+and+type+IV+collagenase.">Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.</a> Journal of Investigative Dermatology. 93 (2), 287-290 (1989).</li><li>Bhatnagar, B. S., Bogner, R. H., Pikal, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+stability+during+freezing:+separation+of+stresses+and+mechanisms+of+protein+stabilization.">Protein stability during freezing: separation of stresses and mechanisms of protein stabilization.</a> Pharmaceutical Development and Technology. 12 (5), 505-523 (2007).</li><li>McClain, A. K., McCarrel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> BMC Veterinary Research. 15 (1), 292 (2019).</li><li>Tamhane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+amniotic+membrane+transplantation+as+an+adjunct+to+medical+therapy+as+compared+with+medical+therapy+alone+in+acute+ocular+burns.">Evaluation of amniotic membrane transplantation as an adjunct to medical therapy as compared with medical therapy alone in acute ocular burns.</a> Ophthalmology. 112 (11), 1963-1969 (2005).</li><li>Shtein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum-based+eye+drops+for+treatment+of+ocular+surface+disease.">Autologous serum-based eye drops for treatment of ocular surface disease.</a> Ophthalmology. 127 (1), 128-133 (2020).</li><li>Shahriari, H., Tokhmehchi, F., Reza, M., Hashemi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effect+of+amniotic+membrane+suspension+and+autologous+serum+on+alkaline+corneal+epithelial+wound+healing+in+the+rabbit+model.">Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model.</a> Cornea. 27 (10), 1148-1150 (2008).</li><li>Schuerch, K., Baeriswyl, A., Frueh, B., Tappeiner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+amniotic+membrane+transplantation+for+the+treatment+of+corneal+ulcers.">Efficacy of amniotic membrane transplantation for the treatment of corneal ulcers.</a> Cornea. 39 (4), 479-483 (2019).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+transplantation+for+persistent+corneal+ulcers+and+perforations+in+acute+fungal+keratitis.">Amniotic membrane transplantation for persistent corneal ulcers and perforations in acute fungal keratitis.</a> Cornea. 25 (5), 564-572 (2006).</li><li>Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+effectiveness+between+amniotic+membrane+homogenate+and+transplanted+amniotic+membrane+in+healing+corneal+damage+in+a+rabbit+model.">A comparison of the effectiveness between amniotic membrane homogenate and transplanted amniotic membrane in healing corneal damage in a rabbit model.</a> Acta Ophthalmologica. 89 (4), 315-319 (2011).</li><li>Sabater, A., Perez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+use+for+management+of+corneal+limbal+stem+cell+deficiency.">Amniotic membrane use for management of corneal limbal stem cell deficiency.</a> Current Opinion in Ophthalmology. 28 (4), 363-369 (2017).</li><li>Ahmad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysis+of+bovine+skin,+its+endogenous+proteases,+protease+inhibitors+and+their+effects+on+quality+characteristics+of+extracted+gelatin.">Autolysis of bovine skin, its endogenous proteases, protease inhibitors and their effects on quality characteristics of extracted gelatin.</a> Food Chemistry. 265, 1-8 (2018).</li><li>Mullegama, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+extraction+from+human+biological+samples.">Nucleic acid extraction from human biological samples.</a> Methods in Molecular Biology. 1897, 359-383 (2019).</li><li>Skog, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+enzymatic+digestion+on+cultured+epithelial+autografts.">The effect of enzymatic digestion on cultured epithelial autografts.</a> Cell Transplantation. 28 (5), 638-644 (2019).</li></ol>

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?...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 N H2SO4 stop solution</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Petri dish</td>
			<td>Symlaboratorios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 G Needle (1.2 mm x 40 mm)</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>305211</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL plastic syringe&#160;</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>302562</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>SARSTEDT</td>
			<td>821581</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Amniotic membrane</td>
			<td>Instituto de Oftalmologia Conde de Valenciana Amnion Bank</td>
			<td></td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Bausch + Lomb</td>
			<td>BSS-403802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender&#160;</td>
			<td>BioRender</td>
			<td></td>
			<td>figures design&#160;</td>
		</tr>
		<tr>
			<td>Compact Rocker</td>
			<td>BioRad</td>
			<td>970822DD</td>
			<td>Mod. 5202SD-BIO</td>
		</tr>
		<tr>
			<td>complete, EDTA-free, Protease inhibitor 
			cocktail tablets</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Daiggner vortex Genie 2</td>
			<td>A.Daigger &#38; Co. , INC</td>
			<td>22220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate spectrometer</td>
			<td>Thermo Labsystems&#160;</td>
			<td>35401106</td>
			<td>Multiscan</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism&#160;</td>
			<td>GraphPad Software, Inc</td>
			<td>version 9</td>
			<td>statistical analysis and graphic program&#160;</td>
		</tr>
		<tr>
			<td>Human lumican DuoSet ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>DY2846-05</td>
			<td>includes human Lumican capture antibody</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Forma Scientific&#160;</td>
			<td>3326 S/N 36481-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light Microscope</td>
			<td>Olympus&#160;</td>
			<td>6A13921</td>
			<td>to confirm de-epithelialization&#160; Mod.CK2</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Forma Scientific&#160;</td>
			<td>14753-567</td>
			<td>Mod.1184</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Mod. Biocan 20</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>R&#38;D Systems</td>
			<td>DY006</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Modified Lowry Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23240</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealers</td>
			<td>R&#38;D Systems</td>
			<td>DY992</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent diluent</td>
			<td>R&#38;D Systems</td>
			<td>DY995</td>
			<td>1% BSA in PBS, pH 7.2-7.4, 0.2 &#956;m filtered</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>centurion scientific Ltd&#160;</td>
			<td>15877</td>
			<td>Mod. K2015R</td>
		</tr>
		<tr>
			<td>Rubber policeman cell scraper</td>
			<td>NEST</td>
			<td>710001</td>
			<td>for mechanical de-epithelialization</td>
		</tr>
		<tr>
			<td>Scalpel knife</td>
			<td>Braun</td>
			<td>BB521</td>
			<td>No. 10 or 21</td>
		</tr>
		<tr>
			<td>Streptavidin-HRP 40-fold concentrated&#160;</td>
			<td>R&#38;D Systems</td>
			<td>part 893975</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate tetramethylbenzidine (TMB) solution</td>
			<td>R&#38;D Systems</td>
			<td>DY999</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezers</td>
			<td>Invent Germany</td>
			<td>6b</td>
			<td>inox&#160;</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>R&#38;D Systems</td>
			<td>WA126</td>
			<td>0.05% Tween 20 in PBS, pH 7.2-7.4</td>
		</tr>
	</tbody>
</table>

lumican extraction from amniotic membrane and determination of its storage temperature

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。

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?...

Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

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。

从羊膜中提取Lumican及其储存温度的测定

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

该方法旨在展示一种简单的蛋白质提取技术，可用于获得丰富的蛋白质和其他因素。该技术可用于提取羊膜中存在的其他因子和蛋白质，也可以从其他来源（例如动物和植物组织）获得蛋白质。首先解冻从羊膜库获得的100毫克羊膜。解冻后，将10毫升无菌平衡盐溶液加入培养皿中，并洗涤膜两分钟。然后，将用过的平衡盐溶液放入烧杯中并重复该过程，直到培养皿中看不到甘油培养基的痕迹。接下来，向膜中加入10毫升分散酶II，并在37摄氏度和5%二氧化碳下孵育30分钟。在孵育结束时，用橡胶警察细胞刮刀进行机械去上皮化。然后，在倒置显微镜下观察膜以确认去上皮化。接下来，如前所述，用无菌平衡盐溶液洗涤去上皮化的羊膜，并将其放入两毫升微量离心管中。然后，将装有膜的管浸入液氮中40分钟。用液氮冷冻后，在研钵中手动研磨膜两到三分钟，在零下85摄氏度下预冷，直到得到细粉。接下来，加入2.5毫升蛋白酶抑制剂溶液并溶解细粉状膜。在手术刀的帮助下清洁砂浆壁，并将混合物转移到五毫升的管中。涡旋试管30秒。然后，首先在4摄氏度下以34 G离心20分钟，然后立即在4摄氏度下以3， 360 G再次离心20分钟，使组织混合物均质化。在离心结束时，将上清液（即羊膜提取物）收集在新管中。然后，在不同的两毫升微量离心管中等分 0.7 毫升，用于不同的时间点和温度。羊膜提取物中的总蛋白量和发光聚糖浓度受时间和贮藏条件的影响。所有羊膜提取物的基础蛋白浓度相似。然而，在4摄氏度下储存32天和在零下20摄氏度下储存20天的提取物中注意到显着高的蛋白质浓度。羊膜提取物中的Lumican在储存12天的样品中明显高于在4摄氏度和零下20摄氏度下储存30天、20天和6天的样品。这些结果表明，在零下20摄氏度下，达到夜光聚糖最高浓度的适当储存时间和温度为12天。在将冷冻羊膜接地时要格外小心。接地应轻柔，因为它可能会从砂浆中掉出来。需要进一步的研究来确定羊膜提取物lumican在角膜上皮细胞中的作用，并确定Lumican用于角膜再上皮化的理想剂量。

Research

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Medicine

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