Name: lumican extraction from amniotic membrane and determination of its storage temperature
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Tüm deneysel prosedürler Kurumsal Gözden Geçirme Kurulu tarafından onaylanmıştır (Proje No. CEI-2020/06/04). , Chavez-García ve ark.17 tarafından tanımlandığı gibi hazırlanan Instituto de Oftalmologia Conde de Valenciana amniyon bankasından (tanımlanmamış insan deneklerden) elde edilmiştir.
1. Amniyotik membran ekstraktının hazırlanması
<ol><li>Amniyon bankasından 100 mg alın. NOT: Önceki bir rapora göre, 50 mg toplam 10...

<ol>
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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. 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Bu çalışmada, AME'de lumikanın varlığı ve farklı depolama koşulları altındaki stabilitesi ile doğrudan ilişkisi analiz edilmiştir. İlginç bir şekilde, AME'deki toplam protein konsantrasyonu ölçüldüğünde, protein konsantrasyonu depolamadan sonra artmıştır. Kanıtlar, dondurulmuş depodaki protein konsantrasyonunu değiştirebilecek üç mekanizma önermektedir: soğuk denatürasyon, çözünenlerin dondurulmuş konsantrasyonu ve protein yapısının buzla indüklenen kısmi açılması<sup class=...

Kornea etkileri için en çok kullanılan tedavilerden biri amniyotik membran transplantasyonudur; ancak son yıllarda amniyotik dokunun çeşitli bileşenlerinin alternatif ve adjuvan tedaviler olarak kullanılması için yeni öneriler ortaya çıkmıştır. 'nin en çok çalışılan bileşenleri arasında ekstraktından (AME) elde edilenler 1,2,3,4,5,6,7'dir. , antianjiyojenik proteinler, interlökinler (IL), metalloproteinazların doku inhibitörleri (TIMP'ler), nötrofil hücre dışı tuzaklarını inhibe eden TSG-6'nın aracılık ettiği anti-enflamatuar proteinler, büyüme faktörleri: epidermal büyüme faktörü (EGF), dönüştürücü büyüme faktörü (TGF) (alfa ve beta), keratinosit büyüme faktörü (KGF), hepatosit büyüme faktörü (HGF) ve kollajen fibrillogenezidüzenleyerek kornea saydamlığını koruyan lumikan gibi çoklu çözünür faktörler içerir 1, 2,3,4,5,6,7,8,9.
Lumikan, kornea stroma matriksindeki interstisyel kollajenazın ana hücre dışı bileşenlerinden biri olan, kollajen liflerinin organize edilmesinden ve kornea saydamlığının korunmasından sorumlu olan küçük lösin bakımından zengin bir proteoglikan (SLRP) 4,10,11'dir. Proteoglikanlar, hücre dışı matriksteki (ECM) moleküllerdir ve hücre içi sinyalizasyonun gerçekleştirilmesinde ve hücre içi homeostazın korunmasında ana moleküllerdir12. ECM proteinlerinin yara iyileşmesi sırasında proliferasyon, farklılaşma ve göçün hücresel süreçlerini yönlendirdiği bildirilmiştir11.
Kanıtlar, lumikanın kornea reepitelizasyonu sürecine olası katılımını göstermektedir. Saika ve ark., bir çalışmada, kornea yaralanmasından sonra, kornea keratositlerinde lumikanın ilk 8 saat ile yaralanmadan sonraki 3 gün arasında tespit edilebileceğini göstermiştir. İkinci ve üçüncü günde en yüksek lumikan konsantrasyonunu sunan bu proteoglikan daha sonra yedinci gün13'te tespit edilemez. Bu veriler, lumikanın kornea reepitelizasyon sürecinin aktivasyonuna katılımını göstermektedir. Öte yandan, başka bir çalışmada, lumikan yokluğunun yeniden epitelizasyonu geciktirdiği; İlginçtir ki, lumikan eklenmesi yeniden epitelizasyon sürecini hızlandırabilir 4,11,13. Benzer şekilde, yakın tarihli bir çalışma, lumikanın kornea limbus fibroblastlarının14'ünün enflamatuar fonksiyonlarını modüle edebildiğini bildirmiştir, bu da lumikanın enflamatuar, antifibrotik ve yeniden epitelleştirici yanıtın bir modülatörü olarak rol oynadığını düşündürmektedir. Benzer şekilde, lumikan, Fas-FasL gibi sinyal molekülleriyle etkileşime girerek kornea tepkisini modüle edebilir. Ayrıca, nakavt bir Lum-/- fare modelinde lumikanın bulunmaması, lumikan sinyalinin eksikliğinin yeterli kornea onarımını engellediğini göstermiştir15.
Öncelikle, bu yöntem lumikanı'den çıkarmanın uygulanabilir ve ulaşılabilir bir yolunu göstermeyi amaçlamaktadır. Bu avantajlı lumikan ekstraksiyonu yöntemiyle, benzer konsantrasyonlarda protein elde etmek, işlem süresini azaltmak ve önceki çalışmalara kıyasla araştırmacılar için daha uygun hale getirmek mümkündür16. Ayrıca, bu AME lumikanı kornea onarımı ve yeniden epitelizasyon işlemleri için bir adjuvan olarak kullanılabilir.

<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

Lumikan, insan amniyotik membranında () kornea epitelizasyonunu ve kollajen liflerinin organizasyonunu teşvik eden, kornea saydamlığını koruyan küçük bir lösin bakımından zengin proteoglikandır. Bu çalışmada, lumikan elde etmek için'den protein ekstraksiyonu için bir yöntem önerilmiştir. Ek olarak, farklı sıcaklık ve zaman periyotlarında depolanan ekstraktındaki (AME) lumikanın stabilitesi değerlendirilir. 100 mg çözüldü ve mekanik olarak epitelden arındırıldı. Deepitelize edilmiş, proteaz inhibitörleri ile 2.5 mL salin tamponu ile çözündürülen ve protein ekstraksiyonu için santrifüj edilen ince bir toz elde edilene kadar donduruldu ve ezildi. Süpernatant toplandı ve 6, 12, 20 ve 32 gün boyunca -20 ° C, 4 ° C ve oda sıcaklığında (RT) saklandı. Daha sonra, lumican her AME'de nicelleştirildi. Bu teknik,'den lumikan ekstraksiyonu için erişilebilir ve edinilebilir bir protokol sağlar. Lumikan konsantrasyonu depolama süresi ve sıcaklık koşullarından etkilendi. -20 ° C ve 4 ° C'de depolanan 12 günlük AME'deki Lumikan, diğer AME'lerden önemli ölçüde daha yüksekti. Bu lumican ekstraksiyonu, tedaviler ve farmasötik çözeltiler geliştirmek için yararlı olabilir. AME lumikan'ın reepitelizasyon ve yara iyileşme sürecindeki kullanımlarını belirlemek için daha ileri çalışmalara ihtiyaç vardır.

Sonuçlar standart sapmanın (SD) ortalama değeri ± olarak raporlanır. Öğrencinin t-testleri ve varyans analizi (ANOVA) yapıldı. 0.05 < P değerleri istatistiksel olarak anlamlı kabul edildi. İstatistiksel analiz, istatistik yazılımı kullanılarak gerçekleştirilmiştir (bakınız Malzeme Tablosu).
AME'deki toplam protein miktarı zaman ve depolama koşullarından etkilenmiştir. Bazal protein konsantrasyonu tüm AME arasında benzerdi; toplam pr...

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

Mevcut protokol, lumikanın amniyotik membrandan () ekstraksiyonunu ve depolama koşullarını, proteinlerini ve lumikan konsantrasyonunu ölçmek için -20 ° C, 4 ° C'de ekstraktı (AME) ve 6, 12, 20 ve 32 gün boyunca oda sıcaklığında (RT) olarak tanımlamaktadır.

Amniyotik membrandan Lumikan ekstraksiyonu ve depolama sıcaklığının belirlenmesi

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

Bu yöntem, bol miktarda protein ve diğer faktörlerin elde edilmesi için yararlı olan protein ekstraksiyonu için basit bir teknik göstermeyi amaçlamaktadır. Bu teknik, amniyotik membranda bulunan diğer faktörleri ve proteinleri çıkarmak ve ayrıca hayvanlar ve bitkisel dokular gibi diğer kaynaklardan proteinler elde etmek için kullanılabilir. Amniyon bankasından elde edilen 100 miligram amniyotik zarı çözerek başlayın.Çözüldükten sonra, bir Petri kabına 10 mililitre steril dengeli tuz çözeltisi ekleyin ve membranı iki dakika yıkayın. Daha sonra, kullanılan dengeli tuz çözeltisini bir beher içine çıkarın ve Petri kabında gliserol ortamı izi görünmeyene kadar işlemi tekrarlayın. Daha sonra, membrana 10 mililitre dispase II ekleyin ve 30 dakika boyunca 37 santigrat derece ve% 5 karbondioksitte inkübe edin.Kuluçkanın sonunda, bir lastik polis hücresi kazıyıcı ile mekanik bir deepitelizasyon gerçekleştirin. Ardından, deepitelizasyonu doğrulamak için membranı ters çevrilmiş bir mikroskop altında görselleştirin. Daha sonra, epitelden arındırılmış amniyotik membranı daha önce gösterildiği gibi steril dengeli tuz çözeltisi ile yıkayın ve iki mililitrelik bir mikrosantrifüj tüpüne yerleştirin.Ardından, membranı içeren tüpü 40 dakika boyunca sıvı azot içine batırın. Sıvı azotla dondurma işleminden sonra, ince bir toz elde edilene kadar membranı eksi 85 santigrat derecede önceden soğutulmuş bir harçta iki ila üç dakika manuel olarak öğütün. Daha sonra, 2.5 mililitre proteaz inhibitörü çözeltisi ekleyin ve ince toz haline getirilmiş membranı çözün.Harç duvarlarını bir neşter bıçağı yardımıyla temizleyin ve karışımı beş mililitrelik bir tüpe aktarın. Tüpü 30 saniye boyunca vorteksleyin. Daha sonra, doku karışımını önce dört santigrat derecede 20 dakika boyunca 34 G'de santrifüj yaparak ve hemen dört santigrat derecede 20 dakika boyunca 3.360 G'de tekrar santrifüj ederek homojenize edin.Santrifüjlemenin sonunda, amniyotik membran ekstresi olan süpernatanı yeni bir tüpte toplayın. Daha sonra, çeşitli zaman noktaları ve sıcaklıklar için farklı bir iki mililitrelik mikrosantrifüj tüpünde 0.7 mililitre aliquot. Amniyotik membran ekstraktındaki toplam protein miktarı ve lumikan konsantrasyonu zaman ve depolama koşullarından etkilenmiştir.Bazal protein konsantrasyonu tüm amniyotik membran ekstraktları arasında benzerdi. Bununla birlikte, dört santigrat derecede 32 gün ve eksi 20 santigrat derecede 20 gün boyunca depolanan ekstraktta önemli ölçüde yüksek bir protein konsantrasyonu kaydedildi. Amniyotik membran ekstraktındaki Lumikan, 12 günlük bir süre boyunca saklanan numunede, dört ve eksi 20 santigrat derecede 30, 20 ve altı gün boyunca saklanan örneklerle karşılaştırıldığında anlamlı derecede daha yüksekti.Bu sonuçlar, en yüksek lumikan konsantrasyonunu elde etmek için uygun depolama süresinin ve sıcaklığının eksi 20 santigrat derecede 12 gün olduğunu göstermiştir. Dondurulmuş amniyotik membranı topraklarken azami özen gösterin. Topraklama, harçtan düşebileceği için yumuşak olmalıdır.Amniyotik membran ekstraktı lumikanının kornea epitel hücrelerindeki rolünü belirlemek ve kornea reepitelizasyonu için ideal lumikan dozunu belirlemek için daha ileri çalışmalara ihtiyaç vardır.

lumican-extraction-from-amniotic-membrane-determination-its-storage

Research

JoVE Journal

Medicine

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermic Tekniği<em&gt; Ex vivo</em&gt; Karaciğer Depolama, Değerlendirme Perfüzyon ve Marjinal Karaciğer Greft Onarımı

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Tıp

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Prematüre Modelleme Ensefalopati Sıçanlarda İçi amniyotik Lipopolisakkarid ile Prenatal Hipoksi-iskemi kullanma

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Using an Ingestible Telemetric Temperature Pill to Assess Gastrointestinal Temperature During Exercise

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of heat-related disorders. Therefore, accurate measurement of Tc during exercise is of great importance, especially in athletes who have to perform in challenging ambient conditions. In the current literature a number of methods have been described to measure the Tc (esophageal, external tympanic membrane, mouth or rectum). However, these methods are suboptimal to measure Tc during exercise since they are invasive, have a slow response or are influenced by environmental conditions. Studies described the use of an ingestible telemetric temperature pill as a reliable and valid method to assess gastrointestinal temperature (Tgi), which is a representative measurement of Tc. Therefore, the goal of this study was to provide a detailed description of the measurement of Tgi using an ingestible telemetric temperature pill. This study addresses important methodological factors that must be taken into account for an accurate measurement. It is recommended to read the instructions carefully in order to ensure that the ingestible telemetric temperature pill is a reliable method to assess Tgi at rest and during exercise.

This study describes an accurate, reliable and non-invasive technique to continuously measure gastrointestinal temperature during exercise. The ingestible telemetric temperature pill is suitable to measure gastrointestinal temperature in laboratory settings as well as in field based settings.

Sindirilebilir bir Telemetrik Sıcaklık hapı kullanma Egzersiz sırasında Gastrointestinal Sıcaklık değerlendirin

Exercise results in an increase in core body temperature (Tc), which may reduce exercise performance and eventually can lead to the development of ...

Esophageal Heat Transfer for Patient Temperature Control and Targeted Temperature Management

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often performed for neuroprotection after ischemic insult (e.g. hemorrhagic stroke, subarachnoid hemorrhage, cardiac arrest, or other hypoxic injury). Cooling from febrile states treats fever and reduces the negative effects of hyperthermia on injured neurons. Patients are warmed in the operating room to prevent inadvertent perioperative hypothermia, which is known to cause increased blood loss, wound infections, and myocardial injury, while also prolonging recovery time. There are many reported approaches for temperature management, including improvised methods that repurpose standard supplies (e.g., ice, chilled saline, fans, blankets) but more sophisticated technologies designed for temperature management are typically more successful in delivering an optimized protocol. Over the last decade, advanced technologies have developed around two heat transfer methods: surface devices (water blankets, forced-air warmers) or intravascular devices (sterile catheters requiring vascular placement). Recently, a novel device became available that is placed in the esophagus, analogous to a standard orogastric tube, that provides efficient heat transfer through the patient&#39;s core. The device connects to existing heat exchange units to allow automatic patient temperature management via a servo mechanism, using patient temperature from standard temperature sensors (rectal, Foley, or other core temperature sensors) as the input variable. This approach eliminates vascular placement complications (deep venous thrombosis, central line associated bloodstream infection), reduces obstruction to patient access, and causes less shivering when compared to surface approaches. Published data have also shown a high degree of accuracy and maintenance of target temperature using the esophageal approach to temperature management. Therefore, the purpose of this method is to provide a low-risk alternative method for controlling patient temperature in critical care settings.

This study presents a novel method to provide efficient patient temperature control for cooling or warming patients. A single use, triple lumen device is placed into the esophagus, analogous to a standard orogastric tube, and connects to existing heat exchange units to perform automatic patient temperature management.

Özofagus ısı transferi hasta sıcaklık kontrolü ve hedeflenen sıcaklık yönetimi için

Controlling patient temperature is important for a wide variety of clinical conditions. Cooling to normal or below normal body temperature is often ...

Microelectrode Impalement Method to Record Membrane Potential from a Cannulated Middle Cerebral Artery

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ion channels and electrogenic pumps that regulate Vm in disease conditions could potentially alter Vm, vascular tone, and blood flow. Thus, a basic understanding of electrophysiology and the methods necessary to accurately record Vm in healthy and diseased states are essential. This method will allow modulating Vm using different pharmacological agents to restore Vm. Although there are several methods, each with its advantages and disadvantages, this article provides protocols to record Vm from cannulated resistance vessels such as the middle cerebral artery using the microelectrode impalement method. Middle cerebral arteries are allowed to gain myogenic tone in a myograph chamber, and the vessel wall is impaled using high resistance microelectrodes. The Vm signal is collected through an electrometer, digitized, and analyzed. This method provides an accurate reading of the Vm of a vessel wall without damaging the cells and without changing the membrane resistance.

The primary goal of this article is to provide details of how to record membrane potential (Vm) from the middle cerebral artery using the microelectrode impalement method. The cannulated middle cerebral artery is equilibrated to gain myogenic tone, and the vessel wall is impaled using high resistance microelectrodes.

Mikroelektrot Impalement yöntemi bir kanüle orta serebral arter membran potansiyelini kaydetmek için

Membrane potential (Vm) of vascular smooth muscle cells determines vessel tone and thus blood flow to an organ. Changes in the expression and function of ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Nitrik Oksit ve Türevlerinin Otoksidasyon ve Nitroslu Bileşiklerden Tespiti için Kemilüminesans Bazlı Testler

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 &#176;C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

Noninvaziv Mikrodiyaliz Tekniği ile Sıçan Kalbinden Dinamik Sürekli Kan Ekstraksiyonu

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

Modifiye edilmiş bir kit kullanarak ekonomik diatom DNA ekstraksiyonu

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

Yazar Spotlight: Adli Boğulma Muayenesi için Diatom Testi 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

Su Örneklerinden ve Dokulardan Diatom DNA'nın Ekstraksiyonu

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...