Kornea Epitel Yara İyileşmesi sırasında Isı Şoku Protein 27 Fonksiyon RNA Girişim tabanlı İncelenmesi
Summary
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.
Abstract
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.
Introduction
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'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'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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Biological safety cabinet | CHC LAB Co.Ltd, Daejeon, Republic of Korea | CHC-777A2-06 | Class II, Type A2 |
| Stealth RNAi™ siRNA | Thermo Fisher Scientific, Inc., Waltham, MA | RNAi siRNA; scrambled control-siRNA and HSP27-specific siRNA | |
| BEGMTM | Lonza, Inc., Walkersville, MD | CC-3171, CC4175 | Bronchial epithelium growth medium |
| Protease inhibitor | Sigma-Aldrich, Inc., St. Louis, MO | P8340 ,P7626 | 1 uM Pepstatin A, 1 uM Leupetin, 0.1 uM Aprotin |
| Bradford protein assay | Bio-Rad Laboratories, Hercules, CA | #500-0001 | Bradford protein assay |
| Nitrocellulose filters | Amersham, Little Chalfont, UK | RPN3032D | Western blotting membrane |
| Non-phosphorylated HSP27 | Abcam Inc., Cambridge, MA | ab12351 | 1:1000 dilution (Total HSP27) |
| Phosphorylated HSP27 (Ser85) | Abcam Inc., Cambridge, MA | ab5594 | 1: 1000 dilution HSP27 was phosphorylated at Ser85 |
| Lipofectamine® RNAiMAX reagent | Invitrogen, Carlsbad, CA | 13-778-075 | Transfection reagent |
| Phosphorylated Akt (Ser473) | Cell Signaling Technology, Danvers, MA | No. 4060 | 1: 1000 dilution Akt was phosphorylated at Ser473 (cell survival marker) |
| Non-phosphorylated Akt | Cell Signaling Technology, Danvers, MA | No. 4061 | 1:1000 dilution (Total Akt) |
| Bcl-2-associated X protein | Cell Signaling Technology, Danvers, MA | No. 4062 | 1: 1000 (anti-apoptotic protein marker) |
| GAPDH | Santa Cruz Biotechnology, Santa Cruz, CA | No. 4063 | 1:1000 loading control marker (house keeping gene) |
| Horseradish peroxidase-conjugated goat anti-rabbit antibodies | Thermo Fisher Scientific, Inc., Waltham, MA | NCI1460KR | 1:10000 dilution |
| OPTI-MEM | Invitrogen, Carlsbad, CA | 31985 | reduced serum medium for transfection |
| Image analysis software | Olympus, Inc., Tokyo, Japan | Image-Pro Plus 5.0 | |
| Skimed milk powder | Carl Roth GmbH + Co. KG, Karlstruhe, Germany | T145.2 | |
| Tris | Amresco LCC, Inc. Solon, OH | No-0497 | |
| Sodium Chloride | Amresco LCC, Inc. Solon, OH | No-0241 | |
| Six well culture plate | Thermo Fisher Scientific, Inc., Waltham, MA | 140675 | 35.00 mm diameter / well |
| 24-well culuture dish | Thermo Fisher Scientific, Inc., Waltham, MA | 142475 | |
| Orbital shaker | N-Bioteck, Inc., Seoul, South Korea | NB1015 | |
| Bovine serum albumin | Santa Cruz Biotechnology, Santa Cruz, CA | sc-2323 | |
| BDFACSCantoTM II | BD Biosciences, Franklin Lakes, NJ | Flow cytometry | |
| X-Ray Film | Kodak, Rochester, NY | Medical X-Ray Cassette with Green 400 Screen | |
| western blotting luminol reagent | Santa Cruz Biotechnology, Santa Cruz, CA | sc-2048 | |
| FITC Annexin V Apoptosis Detection Kit I | BD Biosciences, Franklin Lakes, NJ | 556547 |
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