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.

שורת תאים 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות 10 6 תאים אפיתל הקרנית אדם הנציח טלומרז (HCECs) בצלחת 6-היטב (צפיפות: 1039.9 תא / מ&quot;מ 2) בתוך 37 ° C חממה עם אווירה 5% CO 2 באמצעות מדיום הגידול האפיתל הסימפונות (BEGM) עד הם מגיעים למפגש 95%. </li></ol> 2. ניתוח כתם המערבי לאחר יצירת פצעים Scratch אפיתל <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Streak קצה פיפטה 200 μl סטרילי על פני באר HCECs תרבותי ומחוברות ארבע פעמים לכל צלחת בארון בטיחות ביולוגית (Class II, A2 סוג) דגירה HCECs בנפרד 37 ° C חממה עם אווירה 5% CO 2 עבור 1, 5, 10, 30, 60, ו -120 דקות. </li><li style=";text-align:right;direction:rtl"> השתמש צלחת אחת 6-היטב מזה שישה מדגמים שונים בהתאם לזמן הדגירה לאחר פציעת אפיתל קרנית. </li><li style=";text-align:right;direction:rtl"> לשטוף monolayers HCEC נפצע שלוש פעמים עם 1x PBS ולאחר מכן להוסיף 2.0 מ&quot;ל BEGM היטב כל אחד. </li><li style=";text-align:right;direction:rtl"> ניתוק HCECs באמצעות 2מ&quot;ל 0.25% טריפסין-ethylenediaminetetraacetic חומצה (EDTA) לכל היטב במשך 5 דקות, צנטריפוגות ב 900 XG במשך 5 דקות בתוך שפופרת 15 מ&quot;ל, ואת לשאוב טריפסין-EDTA בעזרת פיפטה 1 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> להשעות HCECs עם 1 מ&quot;ל 1x PBS ולהעבירם צינור 1.5 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> HCECs צנטריפוגה ב XG 10,000 במשך 15 שניות ו PBS 1x לשאוב בעזרת פיפטה 1 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> Resuspend HCECs ב 100 חיץ μl קרים כקרח תמוגה (10 מ&quot;מ טריס, 10 mM NaCl, 2 מ&quot;מ EDTA, 25 מ&quot;מ NaF, 2 מ&quot;מ Na 3 VO 4, 1 מ&quot;מ פלואוריד phenylmethanesulfonyl [PMSF], מעכבי פרוטאז [1 מיקרומטר pepstatin A, 1 מיקרומטר leupeptin, ו -0.1 מיקרומטר aprotinin] ו -0.5% Triton X-100 [pH 7]) ומערבבים אותם היטב. </li><li style=";text-align:right;direction:rtl"> דגירה תאים עבור 30 דקות על הקרח כדי לגרום תמוגה התא. </li><li style=";text-align:right;direction:rtl"> lysates צנטריפוגה ב 4 ° C. ב XG 10,000 במשך 15 דקות, ולאחר מכן להעביר supernatants 1.5 מ&quot;ל צינורות טרי (90 aliquots μl) ולאחסן אותם ב -80 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לקבוע ריכוזי חלבון סך של lysates התא באמצעות יחסי ציבור ברדפורדassay otein. 9 </li><li style=";text-align:right;direction:rtl"> חלבונים הכולל תא טען 30 מיקרוגרם לג&#39;ל acrylamide 10% או 12% עבור ג&#39;ל אלקטרופורזה סולפט-polyacrylamide dodecyl נתרן (SDS-PAGE), ולאחר מכן electrophoretically העברת מופרדים להקות חלבון מסנני nitrocellulose עם זרם 200 מיליאמפר עבור שעה 1 ב 4 ° C להשתמש מבחני כתם מערביים. </li><li style=";text-align:right;direction:rtl"> ממברנות מסנן nitrocellulose בלוק עם 5% חלב רזה מלוחים טריס שנאגרו עם Tween 20 (TBST) במשך שעה 1, להוסיף נוגדנים polyclonal ארנב העיקרי נגד HSP27 הלא פוספורילציה (1: 1,000 דילול) או נוגדנים polyclonal ארנב העיקרי נגד HSP27 פוספורילציה (1 דילול 1,000) ב אלבומין 5% שור (BSA), ו דגירה ממברנות לילה ב 4 ° C על שייקר:. </li><li style=";text-align:right;direction:rtl"> זיהוי להקות immunoreactive באמצעות נוגדנים נגד ארנב peroxidase מצומדות חזרת עיזים (1: 10,000 דילול) ב 5% BSA לאחר הכביסה במשך 10 דקות 3 פעמים עם TBST. </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה של ריאגנטים לומינול המערבי סופג (6-7 מ &#39;l לכל 10 ס&quot;מ × קרום 5 ס&quot;מ) עבור 1 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הסר את הקרום מפתרון מגיב, להסיר נוזל עודף עם מגבה סופגת, ומניח מגן יריעת פלסטיק. </li><li style=";text-align:right;direction:rtl"> עבודה בחדר חשוך עם אור בטוח, להציב קרום מכוסה קלטת סרט עם צד חלבון הפונה כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> מניחים הסרט X-ray על גבי הממברנה ולחשוף דקות 1. </li></ol> 3. siRNA Transfection Assay 10 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> HCECs תרבות ב 5 × 10 5 תאים / גם בצלחת 6-היטב בתוך 37 ° C חממה עם אווירה 5% CO 2 באמצעות BEGM עד שהם מגיעים למפגש 95%. </li><li style=";text-align:right;direction:rtl"> לדלל את מגיב transfection (2.5 או 7.5 μl) עם 100 μl בינוני סרום מופחת עבור transfection (גורם לדילול היה 41 או 14.3) ו לפזר את HSP27 הספציפי מקושקש השליטה siRNA ב 100 μl בינוני סרום מופחת ליצור 10 או 50 ננומטר של HSP27 ספציפי מקושקש siRNA שליטה. </li><li style=";text-align:right;direction:rtl"> מערבבים 100 μ; פתרון siRNA l עם מגיב transfection 100 μl מדולל (1: 1 יחס) דגירה את התערובת במשך 15 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> מוסיפים את מתחמי השומנים-siRNA לתאים. אז אחרי 4 תקשורת hr שינוי להשלים BEGM דגירת התאים למשך 2 ימים ב 37 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לנתח תאי transfected על ידי מערבי סופג, כמתואר מ בסעיפים 4.1 ל -4.7 ילדים. </li></ol> 4. Assay כתם המערבי עבור תאים-טרנספקציה siRNA 11 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חלץ HSP27 ספציפיים מקושקשות HCECs שליטה transfected-siRNA בתוך ארון בטיחות ביולוגית באמצעות 100 חיץ תמוגה μl קרים כקרח (10 טריס מ&quot;מ, 10 מ&quot;מ NaCl, 2 מ&quot;מ חומצה ethylenediaminetetraacetic (EDTA), 25 מ&quot;מ NaF, 2 מ&quot;מ Na 3 VO 4, 1 מ&quot;מ פלואוריד phenylmethanesulfonyl (PMSF), מעכבי פרוטאז, ו -0.5% Triton X-100, pH 7). </li><li style=";text-align:right;direction:rtl"> דגירה תאים עבור 30 דקות על הקרח כדי לגרום תמוגה התא. </li><li style=";text-align:right;direction:rtl"> lysates גלולה ב XG 10,000 במשך 15 דקות ולאחר מכן להעביר supernatants אל גיגית 1.5 מיליליטר טריes (90 μl aliquots) ולאחסן אותם ב -80 ° C. </li><li style=";text-align:right;direction:rtl"> לקבוע ריכוזי חלבון של התא lysates באמצעות השיטה ברדפורד. 9 </li><li style=";text-align:right;direction:rtl"> דגימות טענו עם כמויות שווות של חלבוני תא הכוללים על ג&#39;ל acrylamide 10% או 12%, לייחד את הג&#39;ל על SDS-PAGE, ו electrophoretically להעביר את להקות חלבון המופרדות כדי מסנני nitrocellulose עם זרם 200 מיליאמפר עבור שעה 1 ב 4 ° C עד להשתמש מבחני כתם מערביים. </li><li style=";text-align:right;direction:rtl"> ממברנות nitrocellulose מסנן בלוק עם 5% חלב רזה מלוחים טריס שנאגרו עם Tween 20 (TBST) במשך שעה 1, להוסיף נוגדנים ראשוניים נגד HSP27 פוספורילציה הלא פוספורילציה (1: 1,000 דילול), Akt פוספורילציה (1: 1,000 דילול) הלא פוספורילציה Akt (1: 1,000 דילול, לשמש כסמן הישרדות התא), Bcl-2 הקשורים חלבון X (1: 1,000 דילול, משמש חלבון פרו-אפופטוטיים), ו dehydrogenase 3-פוספט glyceraldehyde (GAPDH; 1 : דילול 200, משמש כביקורת טעינה) ב אלבומין 5% שור (BSA), ודגירת ממברנות הלילה ב 4 ° C על שייקר. </li><li style=";text-align:right;direction:rtl"> זיהוי להקות immunoreactive באמצעות נוגדנים נגד ארנב peroxidase מצומדות חזרת עיזים (1: 10,000 דילול) BSA 5% לאחר הכביסה 3 פעמים עם TBST, 10 דקות כל אחד לשטוף. </li><li style=";text-align:right;direction:rtl"> דגירה הממברנה של ריאגנטים לומינול המערבי סופג (6-7 מ&quot;ל לכל 10 ס&quot;מ × 5 ס&quot;מ קרום) עבור 1 דקות בטמפרטורת החדר. </li><li style=";text-align:right;direction:rtl"> הסר את הקרום מפתרון מגיב, להסיר נוזל עודף עם מגבה סופגת, ומניח מגן יריעת פלסטיק. </li><li style=";text-align:right;direction:rtl"> עבודה בחדר חשוך עם אור בטוח, מניח את הקרום המכוסה קלטת סרט עם צד החלבון הפונה כלפי מעלה. </li><li style=";text-align:right;direction:rtl"> מניח סרט X-ray על גבי הקרום ולחשוף דקות 1. </li></ol> 5. נגרמת Scratch ערכת Assay פציעה כיוונית של נדידת תאי 12 <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בארון בטיחות ביולוגית, להפוך פצע ידי גרירת קצה פיפטה סטרילית על פני באר תרבויות ומחוברות שלHSP27 ספציפי-טרנספקציה siRNA או HCECs המלא המקושקש טרנספקציה-siRNA. </li><li style=";text-align:right;direction:rtl"> מיד לאחר פציעה, לשטוף את התאים פעמיים עם בופר פוספט 1x (PBS) ולשמור אותם בתרבויות BEGM באינקובטור 37 מעלות צלזיוס עם אווירה 5% CO 2 למשך 24 שעות לאחר פציעה. </li><li style=";text-align:right;direction:rtl"> לצלם תמונות HCEC באמצעות מיקרוסקופ זקוף בהגדלה 100X 24 שעות לאחר ופצע ולבצע משטחת רקע באמצעות מסנן פקודת תוכנת ניתוח תמונה. </li><li style=";text-align:right;direction:rtl"> באמצעות פקודת המדידות בוחרות, להגדיר השטח של עניין (AOI) עם אותה הצורה מצולע בגודל שיכול לכסות בניצב מקצה לקצה של פצע ראשוני, ולקבוע שלושה AOI שונים באזור הפצוע של כל דגימה. </li><li style=";text-align:right;direction:rtl"> באופן אוטומטי לספור את המספר הסלולרי בכל תחום באמצעות אפשרויות תפריט מדוד רוזן / גודל. </li></ol> 6. ניתוח cytometry זרימה של אפופטוזיס <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> תרבות siRNA-טרנספקציה HSP27 ספציפי ובקרה שלאירנ&quot;א-טרנספקציה HCECs המכילים כל 10 ננומטר siRNA בריכוז של 5 × 10 5 תאים / גם ב 6 צלחות היטב עד התאים מגיעים 95 מפגש% בתוך 37 ° C חממה עם אווירה 5% CO 2 ב BEGM. </li><li style=";text-align:right;direction:rtl"> ניתוק HCECs באמצעות 2 מ&quot;ל 0.25% טריפסין-EDTA לכל טוב במשך 5 דקות, צנטריפוגות ב 900 XG במשך 5 דקות בתוך שפופרת 15 מ&quot;ל, ואת לשאוב טריפסין-EDTA בעזרת פיפטה 1 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> שטפו תאים פעמיים עם קר PBS ולאחר מכן resuspend התאים 1x חיץ מחייב (0.1 M HEPES / NaOH [pH 7.4], 1.4 M NaCl, ו -25 מ&quot;מ CaCl 2) בריכוז של 10 6 תאים / מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> העברת 100 μl ההשעיה תא (1 x 10 5 תאים) לתוך צינור תרבות 5 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> הוסף 5 μl והעמסת מצומדות isothiocyanate V Annexin ו יודיד propidium 5 μl. </li><li style=";text-align:right;direction:rtl"> בעדינות מערבולת התאים דגירה אותם במשך 15 דקות בטמפרטורת החדר בחושך. </li><li style=";text-align:right;direction:rtl"> הוסף 200 μl של חיץ מחייב 1x על צינור אחד ולנתח את התאים על ידי ג זרימהytometer בתוך שעה 1. </li></ol>

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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=Heat+shock+protein+27+phosphorylation+is+involved+in+epithelial+cell+apoptosis+as+well+as+epithelial+migration+during+corneal+epithelial+wound+healing.">Heat shock protein 27 phosphorylation is involved in epithelial cell apoptosis as well as epithelial migration during corneal epithelial wound healing.</a> Exp Eye Res. 118 (1), 36-41 (2014).</li></ol>

יש המחברים שום אינטרסים כלכליים או קניינים כל חומר או שיטות שהוזכר במחקר זה.

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...

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&#39; 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.

<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.

הביטוי של HSP27 פוספורילציה גדל באופן משמעותי ב 5, 10, ו -30 דקות לאחר שריטה ופצע לעומת HCECs unwounded 13. ניתוח כתם המערבי גילה כי הביטוי של פוספורילציה HSP27 ו פוספורילציה Akt הופחתו הן באופן משמעותי, ואילו הביטוי של Bax הוגדל משמעותית HCECs-טרנספקציה siRNA ספציפיים...

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.

RNA הפרעה מבוססת חקירת הפונקציה של חלבון מחממי הלם 27 במהלך הקרנית אפיתל לריפוי פצעים

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

Research

JoVE Journal

Genetics

7.4K Views.  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.

הקמת קלינית רלוונטי<em&gt; Ex Vivo</em&gt; דגם ניתוח קטרקט מוק לחקירת תיקון פצע אפיתל במייקרו-סביבה של ילידים

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

Developmental Biology

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.

הדמיית תאים חיים של גלובוסים שלמים של Ex Vivo באמצעות מחזיק חדשני מודפס בתלת-ממד

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

Biochemistry

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;

חקר התחדשות רקמות ללא צלקות בקרנית אפרוח פצועה עוברית

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.

אפיתל שחיקה הקרנית עם עינית Burr כמודל עבור קרנית ריפוי פצעים

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 ...

Medicine

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 איברים הקרנית מודל תרבות עבור ריפוי פצעים מחקרים

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.

מודל שחיקה אפיתליאלית לחקר ריפוי פצעי הקרנית

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.

מודלים של בעלי חיים של Ex Vivo ו-In Vivo לפציעות מכניות וכימיות של אפיתל הקרנית

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.

תפוקה גבוהה SiRNA ואיתור Chloropicrin ופציעות תאים אפיתל הקרנית מימן פלואוריד-Induced

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

Biology

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.

ריפוי פצעים בקרנית דגי זברה: משחיקה לניתוח הדמיה לסגירת פצעים

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.