Bu çalışma Tıp Seul, Kore Ulsan College Üniversitesi Öğrenci Araştırma Grant (13-14) ve Asan Yaşam Bilimleri Enstitüsü, Seul, Kore bir hibe (2014-464) tarafından desteklenmiştir.

1. Hücre Hattı <ol><li> 6-çukurlu plaka içinde kültürü 10 6 telomeraz-ölümsüzleştirilmiş insan kornea epitel hücreleri (HCECs) bronşiyal epitel büyüme ortamı (Begm) kullanılarak% 5 CO2 atmosferi ile 37 ° C kuluçka makinesi içinde (yoğunluk 1039,9 hücre / mm2 kadar) onlar% 95 izdiham ulaşmak. </li></ol> 2. Western Blot Analizi Epitel Scratch yaralar oluşturma sonra <ol><li> Streak steril 200 ul pipet konfluent kültürlü HCECs bir kuyunun yüzeyi boyunca ucu biyolojik güvenlik kabini çanak başına dört kez (Sınıf II, Tip A2) ve% 5 CO 2 atmosfer ile 37 ° C inkübatör ayrı ayrı HCECs inkübe 1, 5, 10, 30, 60 ve 120 dakika karıştırıldı. </li><li> korneal epitelyal yaralama sonra inkübasyon süresine göre altı farklı numuneler için, bir 6-yuvalı plaka kullanınız. </li><li> 1X PBS ile yaralandı HCEC mono tabakalar üç kez yıkayın ve daha sonra her kuyuya 2.0 mi Begm ekleyin. </li><li> 2 kullanılarak HCECs ayırınmi,% 0.25 tripsin etilendiamintetraasetik 5 dakika boyunca oyuk başına asit (EDTA), 15 ml&#39;lik bir tüp içinde 5 dakika için 900 x g&#39;de santrifüj ve aspire tripsin-EDTA 1 ml pipet kullanarak. </li><li> PBS 1X 1 ml HCECs askıya alma ve bir 1.5 ml&#39;lik tübe transfer edin. </li><li> 15 sn için 10,000 x g&#39;de santrifüjleyin HCECs ve aspirat 1X PBS, 1 ml pipet kullanarak. </li><li> 100 ul buz gibi soğuk lisis tamponunda yeniden süspanse HCECs (10 mM Tris, 10 mM NaCI, 2 mM EDTA, 25 mM NaF, 2 mM Na 3 VO 4, 1 mM fenilmetansülfonil florit [PMSF], proteaz inhibitörleri [pepstatin A 1 uM, 1 uM löpeptin ve 0.1 uM aprotinin] ve% 0.5 Triton X-100 [pH 7]) ve iyice karıştırın. </li><li> Hücre lizizi uyarılması için, buz üzerinde 30 dakika boyunca inkübe hücreleri. </li><li> Santrifüj 15 dakika boyunca 10,000 x g&#39;de 4 ° C&#39;de lizatları, ve sonra taze 1.5 ml tüpler (90 ul tümbölenler) süpernatantlar transferi ve -80 ° C&#39;de saklayın. </li><li> Bradford pr kullanılarak hücre lizatlarının toplam protein konsantrasyonunu belirlemekotein deneyi. 9 </li><li> daha sonra, sodyum dodesil sülfat-poliakrilamid jel elektroforezi (SDS-PAGE) ve bir% 10 ya da% 12 akrilamid jel yükle 30 ug toplam hücre proteinleri elektroforetik transferi 4 ° &#39;de 1 saat süre ile, bir 200 mA akım ile nitroselüloz filtreler protein bantları ayrıldı Cı Western blot deneylerinde kullanım. </li><li> % 5 blok nitroselüloz filtre zarları fosforilatlanmamış Hsp27 karşı birincil tavşan poliklonal antikor ekleyin, 1 saat süre ile, Tween 20 (TBST) Tris-tamponlu tuzlu su içinde yağsız süt (1: 1000 seyrelti) veya fosforile Hsp27 karşı birincil tavşan poliklonal antikoru (1 : 1,000% 5 bovin serum albümini seyreltme) (BSA) ve bir çalkalayıcı üzerinde 4 ° C&#39;de gece boyunca inkübe membranlar. </li><li> TBST ile 10 dakika süre ile 3 kez yıkandıktan sonra,% 5 BSA (10,000 seyreltme 1) yabanturpu peroksidaz-konjuge keçi anti-tavşan antikorları kullanılarak immünoreaktif bantlar algılar. </li><li> Western lekeleme luminol reaktifler membran inkübe (6-7 mOda sıcaklığında 1 dakika boyunca 5 cm&#39;lik membran x 10 cm) başına l. </li><li> reaktif çözeltisinden membran kaldırmak, bir plastik levha koruyucusu aşırı emici bir havlu ile sıvı ve yer çıkarın. </li><li> güvenli bir ışık ile karanlık bir odada çalışan protein tarafı yukarı bakacak şekilde bir film kaseti kaplı membran yerleştirin. </li><li> zarın üzerine X-ışını filmi yerleştirin ve 1 dakika boyunca maruz kalmaktadır. </li></ol> 3. siRNA Transfeksiyon Deneyi 10 <ol><li> Kültür 5 x 10 5 hücre HCECs / oyuk bunlar% 95 izdiham ulaşana kadar Begm kullanılarak% 5 CO2 atmosferi ile 37 ° C kuluçka makinesi içinde 6 oyuklu plaka. </li><li> 10 ya da 50 nM oluşturmak için azaltılmış serum orta transfeksiyon (seyreltme faktörü 41 veya 14.3 idi) için 100 ul azaltılmış serum orta transfeksiyon ayıracı (2.5 veya 7.5 ul) sulandırmak ve Hsp27 özel çözülür ve 100 ul kontrol siRNA şifreli Hsp27 özgü ve kontrol siRNA şifreli. </li><li> 100 mikron Mix100 ul seyreltilmiş transfeksiyon reaktifi ile l siRNA çözeltisi (1: 1 oranında) ve oda sıcaklığında 15 dakika süreyle inkübe karışımı. </li><li> hücrelere siRNA lipid kompleksleri ekleyin. Daha sonra 4 saat değiştirme ortamı sonra 37 ° C&#39;de 2 gün boyunca hücrelerle Begm doldurup kuluçkalanması. </li><li> 4.7 bölümler 4.1 altında tarif edildiği gibi, western blotting ile transfekte edilmiş hücrelerin analiz. </li></ol> SiRNA transfekte hücreleri 11 4. Batı Benek tahlili <ol><li> Hsp27 özgü ekstrakte ve 100 ul buz gibi soğuk lisis tamponu (10 mM Tris, 10 mM NaCI, 2 mM etilendiamintetraasetik asit (EDTA), 25 mM NaF, 2 mM Na 3 VO kullanılarak bir biyolojik güvenlik kabini kontrol siRNA transfekte HCECs karıştırılmış 4, 1 mM fenilmetansülfonil florit (PMSF), proteaz inhibitörleri, ve% 0.5 Triton X-100, pH 7). </li><li> Hücre lizizi uyarılması için, buz üzerinde 30 dakika boyunca inkübe hücreleri. </li><li> 10,000 x g&#39;de Pelet lizatları, 15 dakika ve daha sonra da yeni bir 1.5 ml&#39;lik küvet süpernatantlar aktarmakes (90 ul alikotları) ve -80 ° C&#39;de saklayın. </li><li> Bradford protein deneyi kullanılarak hücre lizatlarının protein konsantrasyonlarını belirlemek. 9 </li><li> % 10 ya da% 12 akrilamid jel üzerinde toplam hücre Proteinlerin eşit miktarları ile Numuneleri, SDS-PAGE jelin maruz ve elektroforetik olarak 4 ° C &#39;de, 1 saat boyunca 200 mA akım ile nitroselüloz filtrelerine Ayrılan protein bantları aktarmak Western blot deneylerinde kullanımı. </li><li> : (1000 sulandırma 1): (1000 sulandırma 1), fosforilatlanmış Akt% 5 blok nitroselüloz filtre zarları fosforillenmiş ve fosforilatlanmamış Hsp27 karşı birincil antikor ekleyin, 1 saat süre ile, Tween 20 (TBST) Tris-tamponlu tuzlu su içinde yağsız süt Akt fosforilatlanmamış (1: 1000 seyreltme, bir hücrelerin hayatta kalma belirteci olarak kullanılır) Bcl-2 ile ilişkili X proteini (1: 1000 seyreltme, bir pro-apoptotik protein olarak kullanılır) ve gliseraldehid 3-fosfat dehidrojenaz (GAPDH 1; :% 5 büyükbaş hayvan serumu albümini bir yükleme kontrolü olarak kullanılan 200 seyreltme) (BSA) vebir çalkalayıcı üzerinde 4 ° C&#39;de gece boyunca inkübe membranlar. </li><li> yıkamadan sonra TBST ile 3 kez 10 dakika her yıkama% 5 BSA (10,000 seyreltme 1) yabanturpu peroksidaz-konjuge keçi anti-tavşan antikorları kullanılarak immünoreaktif bantlar algılar. </li><li> Oda sıcaklığında 1 dakika boyunca Western lekeleme luminol reaktifler membran (6-7 mi başına 10 cm x 5 cm zarı) inkübe edin. </li><li> reaktif çözeltisinden membran kaldırmak, bir plastik levha koruyucusu aşırı emici bir havlu ile sıvı ve yer çıkarın. </li><li> güvenli bir ışık ile karanlık bir odada çalışan protein tarafı yukarı bakacak şekilde bir film kaseti kaplı membran yerleştirin. </li><li> zarın üzerine X-ışını filmi yerleştirin ve 1 dakika boyunca maruz kalmaktadır. </li></ol> 5. Scratch kaynaklı Hücre Göç 12 Yönlü Yaralama Deneyi Değerlendirilmesi <ol><li> Bir biyolojik güvenlik kabini, bir birleşik kültürlerin bir kuyu yüzeyi boyunca steril bir pipet sürükleyerek bir yara yapmakHsp27 özgü siRNA transfekte veya karıştırılmış kontrol siRNA transfekte HCECs. </li><li> Hemen yaralama sonra, yaralama sonrası 24 saat boyunca bir% 5 CO2 atmosferi ile 37 ° C kuluçka makinesi içinde Begm kültürlerinde bunları 1X fosfat tamponlu salin (PBS) ile iki kez yıkama hücreleri ve bakımı. </li><li> 24 saat yaralama sonra 100X büyütmede dik bir mikroskop kullanılarak HCEC görüntüleri fotoğraflamak ve arka plan Filtre kullanarak düzleştirme gerçekleştirmek görüntü analiz yazılımı komut. </li><li> Seç Ölçümler komutunu kullanarak, ilk yara uçtan uca dik kapağı aynı büyüklükteki çokgen şekli ile İlgi (AOI) ait Alan tanımlamak ve her numunenin yaralı alanda üç farklı AOI belirler. </li><li> Otomatik olarak Kont / Boyut Ölçüm menü seçeneklerini kullanarak her alanda hücre sayısını. </li></ol> Apoptoz 6. Akım Sitometri Analizi <ol><li> Kültür Hsp27 özgü siRNA transfekte edilmiş ve kontrol sistemHücreler Begm bir% 5 CO2 atmosferi ile 37 ° C kuluçka makinesi içinde% 95 kaynaşmaya eriştikten kadar / oyuk 6 oyuklu plakalar içerisinde 5 x 10 5 hücre konsantrasyonunda her biri 10 nM siRNA içeren HCECs IRNA-transfekte edildi. </li><li> 1 ml pipet kullanılarak 5, 15 ml tüp içinde dakika ve aspirat tripsin-EDTA 900 x g&#39;de 5 dakika santrifüj için oyuk başına 2 mi% 0.25 tripsin-EDTA kullanılarak HCECs ayırın. </li><li> Soğuk PBS ile iki kez hücreleri yıkanır ve daha sonra 10 6 hücre / ml&#39;lik bir konsantrasyonda bir tampon (0.1 M HEPES / NaOH [pH 7.4], 1.4 M NaCl, ve 25 mM CaCI2) bağlanma 1x hücreleri tekrar süspansiyon. </li><li> 5 ml&#39;lik bir kültürden tüp içine hücre süspansiyonu (1 x 10 5 hücre) ul transfer 100. </li><li> 5 ul flöresin izotiyosiyanat-konjuge Annexin V ve 5 ul propidyum iyodür ekleyin. </li><li> Yavaşça hücreleri girdap ve karanlıkta, oda sıcaklığında 15 dakika boyunca inkübe. </li><li> bir akış c hücreleri her tüpe 1x bağlayıcı tampon 200 ul ekleyin ve analiz1 saat içinde ytometer. </li></ol>

<ol>
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Yazarlar bu çalışmada adı geçen herhangi bir malzeme veya yöntemlerde finansal veya özel çıkarları var.

In this present study, we evaluated the potential role of HSP27 in corneal epithelial wounding using in vitro approaches. The critical steps involved siRNA transfection for HSP27 knock-down to observe the function of HSP27 in cells subjected to stress. Notably, a role for HSP27 was revealed by these experiments in epithelial cell migration and apoptosis during corneal epithelial wound healing. Unlike previous studies10 that used rat HSP27-specific siRNA to transfect vascular smooth muscle cells, we us...

Aynı anda limbusta ve kornea epitel bazal tabakadan hücreler tarafından değiştirilir ise kornea epitel hücreleri (kurulları) sürekli gözyaşı içine dökülürler. 1 Çeşitli dışsal stres CDKÖ apoptozunu ve deskuamasyon uyarabilir. 2 ısı şok proteinleri (HSPler) yüksek ölçüde korunmuş ve molekül büyüklüğüne göre iki aile ayrılabilir. 3 büyük HSP ailesi HSP90, HSP70 ve HSP60, ve daha küçük bir aile Hsp27 içerir içerir. 4. Hsp27 fosforilasyon hücre canlılığı ile önemli bir rol oynadığı bilinmektedir çünkü aktin yeniden bu proteinin rolü hücre göçü için gereklidir. 5-7 Bu nedenle, epitel yara iyileşmesi bir in vitro modelinde MSK göç ve apoptoz Hsp27 fosforilasyonunun potansiyel rolünü test etmek için çalışmıştır. RNA enterferans (RNAi), küçük veya kısa müdahale RNA&#39;lar kullanılarak (siRNA) ge sahiptirpotansiyel verir gibi hem temel ve uygulamalı biyoloji nerated ilgi, ilgi herhangi bir genin ifadesi çaldı aşağı olmak. 8 Burada, MSK yara iyileşmesi ve apoptoz ile Hsp27 katkısını değerlendirmek için Hsp27 özel siRNA kullanılır. hücrelerinde gen knock-down Geleneksel RNAi yöntemler siRNA&#39;lar oluşturmak için monte edilebilir, iki modifiye edilmemiş 21-mer oligonükleotitlerin de dahil olmak üzere, sentetik RNA çiftleri kullanmak. Bu, bu çalışmada kullanılan RNAi siRNA hücreleri transfekte etmek üzere, basit ve oldukça etkili bir yöntem olup, bu reaktif, çeşitli ölümsüz hücre çizgileri ile birlikte çalışır. Bu bu çalışmada, bir çizik kaynaklı yönlü yara deneyi, batı blot, siRNA transfeksiyon deneyinde, immunfloresans yöntemi de dahil olmak üzere, bu analiz için kullanılan yöntemler, göstermek ve akım sitometri.

<table><tbody><tr><td>Biological safety cabinet</td><td>CHC LAB Co.Ltd,&amp;nbsp; Daejeon, Republic of Korea&amp;nbsp;</td><td>CHC-777A2-06</td><td>Class II, Type A2&amp;nbsp;</td></tr><tr><td>Stealth RNAi&amp;trade; siRNA</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td></td><td>RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA</td></tr><tr><td>BEGM&lt;sup&gt;TM&lt;/sup&gt;</td><td>Lonza, Inc., Walkersville, MD</td><td>CC-3171, CC4175</td><td>Bronchial epithelium growth medium&amp;nbsp;</td></tr><tr><td>Protease inhibitor&amp;nbsp;</td><td>Sigma-Aldrich, Inc., St. Louis, MO</td><td>P8340 ,P7626</td><td>1 &amp;#956;M Pepstatin A, 1 &amp;#956;M Leupeptin, 0.1 &amp;#956;M Aprotinin</td></tr><tr><td>Bradford protein assay&amp;nbsp;</td><td>Bio-Rad Laboratories, Hercules, CA</td><td>#500-0001</td><td>Bradford protein assay&amp;nbsp;</td></tr><tr><td>Nitrocellulose filters&amp;nbsp;</td><td>Amersham, Little Chalfont, UK</td><td>RPN3032D</td><td>Western blotting membrane</td></tr><tr><td>Non-phosphorylated HSP27&amp;nbsp;</td><td>Abcam Inc., Cambridge, MA</td><td>ab12351</td><td>1:1,000 dilution (Total HSP27)</td></tr><tr><td>Phosphorylated HSP27 (Ser85)</td><td>Abcam Inc., Cambridge, MA</td><td>ab5594</td><td>1:1,000 dilution HSP27 was phosphorylated at Ser85</td></tr><tr><td>Lipofectamine&lt;sup&gt;&amp;reg; &lt;/sup&gt;RNAiMAX reagent&amp;nbsp;</td><td>Invitrogen, Carlsbad, CA</td><td>13-778-075</td><td>Transfection reagent</td></tr><tr><td>Phosphorylated Akt (Ser473)</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4060</td><td>1:1,000 dilution Akt was phosphorylated at Ser473 (cell survival marker)</td></tr><tr><td>Non-phosphorylated Akt&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4061</td><td>1:1,000 dilution (Total Akt)</td></tr><tr><td>Bcl-2-associated X protein&amp;nbsp;</td><td>Cell Signaling Technology, Danvers, MA</td><td>No. 4062</td><td>1:1,000 (anti-apoptotic protein marker)</td></tr><tr><td>GAPDH</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>No. 4063</td><td>1:1,000 loading control marker (house keeping gene)</td></tr><tr><td>Horseradish peroxidase-conjugated goat anti-rabbit antibodies</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>NCI1460KR</td><td>1:10,000 dilution</td></tr><tr><td>OPTI-MEM</td><td>Invitrogen, Carlsbad, CA</td><td>31985</td><td>reduced serum medium for transfection</td></tr><tr><td>Image analysis software</td><td>Olympus, Inc., Tokyo, Japan</td><td>Image-Pro Plus 5.0</td><td></td></tr><tr><td>Skimed milk powder&amp;nbsp;</td><td>Carl Roth GmbH + Co. KG, Karlstruhe, Germany</td><td>T145.2</td><td></td></tr><tr><td>Tris&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0497</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Amresco LCC, Inc. Solon, OH</td><td>No-0241</td><td></td></tr><tr><td>Six well culture plate</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>140675</td><td>35.00 mm diameter / well</td></tr><tr><td>24-well culuture dish</td><td>Thermo Fisher Scientific, Inc., Waltham, MA</td><td>142475</td><td></td></tr><tr><td>Orbital shaker</td><td>N-Bioteck, Inc., Seoul, South Korea</td><td>NB1015</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2323&amp;nbsp;</td><td></td></tr><tr><td>BDFACSCanto&lt;sup&gt;TM&lt;/sup&gt; II</td><td>BD Biosciences, Franklin Lakes, NJ</td><td></td><td>Flow cytometry</td></tr><tr><td>X-Ray Film</td><td>Kodak, Rochester, NY</td><td></td><td>Medical X-Ray Cassette with Green 400 Screen&amp;nbsp;</td></tr><tr><td>western blotting luminol reagent</td><td>Santa Cruz Biotechnology, Santa Cruz, CA</td><td>sc-2048&amp;nbsp;</td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences, Franklin Lakes, NJ</td><td>556547</td><td></td></tr></tbody></table>

rna interference-based investigation of the function of heat shock protein 27 during corneal epithelial wound healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Fosforile Hsp27 ifadesi anlamlı 5, 10 arttı ve sıfırdan 30 dakika sonra yaralı olmayan HCECs 13 ile karşılaştırıldığında yaralama. Western blot analizi, Bax ekspresyonu anlamlı Hsp27 özgü siRNA transfekte HCECs (Şekil 1A-D) &#39;de artmıştır, oysa fosforile Hsp27 ve fosforile Akt ekspresyon hem de önemli ölçüde azalır olduğunu göstermiştir. Fosforile Hsp27 sentezleme% 30 ve 40 10 nM ve% Hsp27 özgü 50 nM siRNA transfekte edilmiş h...

Watch this Scientific Journal Video about RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing at JoVE.com

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Kornea Epitel Yara İyileşmesi sırasında Isı Şoku Protein 27 Fonksiyon RNA Girişim tabanlı İncelenmesi

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

rna-interference-based-investigation-function-heat-shock-protein-27

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

7.4K Görüntüleme.  Saevit Eye Hospital. Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

Video: RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Bir Klinik İlgili kurulması<em&gt; Ex Vivo</emYerli mikroçevresinin Epitelyal Yara Tamir incelenmesi için&gt; Mock Katarakt Cerrahisi Modeli

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Gelişim Biyolojisi

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation results in calcium mobilization events that propagate from cell to cell, which are essential for initiating cellular motility into the wound bed, promoting efficient wound healing. The Trinkaus-Randall lab has developed a methodology for imaging the corneal wound healing response in ex vivo murine globes in real time. This approach involves enucleating an intact globe from a mouse that has been euthanized per established protocols and immediately incubating the globe with a calcium indicator dye. A counterstain that stains other features of the cell can be applied at this stage to assist with imaging and show cellular landmarks. The protocol worked well with several different live cell dyes used for counterstaining, including SiR actin to stain actin and deep red plasma membrane stain to stain the cell membrane. To examine the response to a wound, the corneal epithelium is injured using a 25 G needle, and the globes are placed in a 3D printed holder. The dimensions of the 3D printed holder are calibrated to ensure immobilization of the globe throughout the duration of the experiment and can be modified to accommodate eyes of different sizes. Live cell imaging of the wound response is performed continuously at various depths throughout the tissue over time using confocal microscopy. This protocol allows us to generate high-resolution, publication-quality images using a 20x air objective on a confocal microscope. Other objectives can also be used for this protocol. It represents a significant improvement in the quality of live cell imaging in ex vivo murine globes and permits the identification of nerves and epithelium.

Live-Cell Imaging of Intact Ex Vivo Globes Using a Novel 3D Printed Holder

The present work describes a novel experimental protocol that utilizes a 3D printed holder to enable high-resolution live cell imaging of enucleated globes. Through this protocol, the cellular calcium signaling activity in wounded corneal epithelium from ex vivo globes can be observed in real time.

Yeni Bir 3D Baskılı Tutucu Kullanarak Bozulmamış Ex Vivo Kürelerin Canlı Hücre Görüntülemesi

Corneal epithelial wound healing is a migratory process initiated by the activation of purinergic receptors expressed on epithelial cells. This activation ...

Biyokimya

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Embriyonik Yaralı Civciv Kornealarında İzsiz Doku Rejenerasyonunun İncelenmesi

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Kornea epitel aşınma oküler Burr ile bir Model olarak kornea için yara iyileşme

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Tıp

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex vivo kornea Organ kültür modeli için yaranın çalışmaları

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

Kornea Yara İyileşmesini İncelemek İçin Epitelyal Aşınma Modeli

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Kornea Epitelinin Mekanik ve Kimyasal Yaralanmaları için Ex Vivo ve In Vivo Hayvan Modelleri

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments for toxicant-induced corneal injury are generally supportive as no specific therapeutics exist to treat these injuries. In the efforts to develop treatments and therapeutics to care for exposure, it can be important to understand the molecular and cellular mechanisms of these injuries. We propose that utilization of high throughput small inhibitory RNA (siRNA) screening can be an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. siRNA are double stranded RNA molecules that are 19-25 nucleotides long and utilize the post-transcriptional gene silencing pathway to degrade mRNA which have homology to the siRNA. The resulting reduction of expression of the specific gene can then be studied in toxicant exposed cells to ascertain the function of that gene in the cellular response to the toxicant. The development and validation of in vitro exposure models and methods for the high throughput screening (HTS) of hydrogen fluoride- (HF) and chloropicrin- (CP) induced ocular injury are presented in this article. Although we selected these two toxicants, our methods are applicable to the study of other toxicants with minor modifications to the toxicant exposure protocol. The SV40 large T antigen immortalized human corneal epithelial cell line SV40-HCEC was selected for study. Cell viability and IL-8 production were selected as endpoints in the screening protocol. Several challenges associated with the development of toxicant exposure and cell culture methods suitable for HTS studies are presented. The establishment of HTS models for these toxicants allows for further studies to better understand the mechanism of injury and to screen for potential therapeutics for chemical ocular injury.

High throughput small inhibitory RNA screening is an important tool that could help to more rapidly elucidate the molecular mechanisms of chemical cornea epithelial injury. Herein, we present the development and validation of exposure models and methods for the high throughput screening of hydrogen fluoride- and chloropicrin-induced cornea epithelial injury.

Yüksek işlem hacmi SiRNA tarama için polis ve hidrojen florür kaynaklı kornea epitel hücre hasarı

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

Biyoloji

Zebrafish Corneal Wound Healing: From Abrasion to Wound Closure Imaging Analysis

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. Indeed, the eye injuries most frequently encountered clinically are those to the cornea. While corneal wound healing has been extensively studied in small mammals (i.e., mice, rats, and rabbits), corneal physiology studies have neglected other species, including zebrafish, despite zebrafish being a classic research model.
This report describes a method of performing a corneal abrasion on zebrafish. The wound is performed in vivo on anesthetized fish using an ocular burr. This method allows for a reproducible epithelial wound, leaving the rest of the eye intact. After abrasion, wound closure is monitored over the course of 3 h, after which the wound is reepithelialized. By using scanning electron microscopy, followed by image processing, the epithelial cell shape, and apical protrusions can be investigated to study the various steps during corneal epithelial wound closure.
The characteristics of the zebrafish model permit study of the epithelial tissue physiology and the collective behavior of the epithelial cells when the tissue is challenged. Furthermore, the use of a model deprived of the influence of the tear film can produce&#160;new answers regarding corneal response to stress. Finally, this model also allows the delineation of the cellular and molecular events involved in any epithelial tissue subjected to a physical wound. This method can be applied to the evaluation of drug effectiveness in preclinical testing.

This protocol focuses on damaging the ocular surface of zebrafish through abrasion to assess the subsequent wound closure at the cellular level. This approach exploits an ocular burr to partly remove the corneal epithelium and uses scanning electron microscopy to track changes in cell morphology during wound closure.

Zebra Balığı Kornea Yara İyileşmesi: Aşınmadan Yara Kapatma Görüntüleme Analizine

As the transparent surface of the eye, the cornea is instrumental for clear sight. Due to its location, this tissue is prone to environmental insults. ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.