RNA הפרעה מבוססת חקירת הפונקציה של חלבון מחממי הלם 27 במהלך הקרנית אפיתל לריפוי פצעים
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
תאי אפיתל קרניים (CECs) הם לשפוך ברציפות לתוך שכבת דמעות, בזמן שהם מוחלפים בו זמנית על ידי תאים מן לימבוס ושכבות הבזליים אפיתל קרניים. 1 לחצים חיצוניים שונים יכולים לגרום אפופטוזיס קלוף של CECs. 2 חלבוני הלם החום (HSPs) שמורים מאוד ניתן לחלק לשתי משפחות לפי גודל מולקולרי. 3 מש' HSP הגדולה כולל Hsp90, HSP70, ו HSP60, והמשפחה הקטנה כולל HSP27. 4 זירחון של HSP27 ידוע לשחק תפקיד חשוב בהישרדות תא והוא נחוץ לצורך נדידת תאים עקב התפקיד של חלבון זה שיפוץ יקטין. 5-7 לכן, ניסינו לבחון את התפקיד הפוטנציאלי של זירחון HSP27 גירת CEC ו אפופטוזיס מודל במבחנה של ריפוי פצע אפיתל.
התערבות RNA (RNAi) באף אחת קטנה או קצר RNAs התערבות (siRNA) יש geעניין nerated בשתי ביולוגיה בסיסית ויישומים, כפי שהוא עשוי לאפשר את הביטוי של כל גן של עניין להיות דפק למטה. 8 בזאת, השתמשנו siRNA ספציפי-HSP27 להעריך את התרומה של HSP27 לריפוי אפופטוזיס פצע CEC. שיטות RNAi מסורתית עבור הגן לדפוק למטה בתאים להשתמש דופלקסים RNA סינתטיים, לרבות שני oligonucleotides 21-mer ללא שינוי, אשר ניתן לקבץ ליצור siRNAs. RNAi siRNA כי השתמשנו במחקר הנוכחי הוא שיטה פשוטה ויעילה מאוד transfect תאים, מגיב זה עובד עם שורות תאים הנציחו שונות. במחקר הנוכחי, אנחנו מדגימים את השיטות בהן השתמשו בניתוח זה, כולל assay הפצע כיוונית הנגרמת מאפס, סופג המערבי, assay transfection siRNA, assay immunofluorescence, cytometry הזרימה.
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
מחקר זה נתמך על ידי מענק מחקר סטודנטים (13-14) של אוניברסיטת אולסן המכללה לרפואה, בסיאול, קוריאה ומענק (2014-464) ממכון אסאן למדעי החיים, בסיאול, קוריאה.
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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