Name: generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells
Uploaded: 2015-02-20T15:00:00
Description: Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

עבודה זו נתמכת על ידי מענקים מהמוסד הקנדי לבריאות מחקר (178,918), Fonds דה המשוכלל והנדיר en סנטה du קוויבק - רשת מחקר בריאות חזון.

הערה: זכר C57BL / 6 עכברים (5-7 שבועות) הם מצ&#39;ארלס ריבר מעבדות International, Inc (St-קבוע, קוויבק, קנדה.) ומניפולציות על פי פרוטוקולים שאושרו על ידי ועדת בעלי החיים Sainte-ג&#39;סטין CHU Care. explants רקמת הסימפונות העכבר מספק מקור טוב לתאי האפיתל הסימפונות ראשיים לחקירת השפעות proapoptotic של LMPs על ?...

<ol>
	<li>Tushuizen, M. E., Diamant, M., Sturk, A., Nieuwland, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+microparticles+in+the+pathogenesis+of+cardiovascular+disease:+friend+or+foe.">Cell-derived microparticles in the pathogenesis of cardiovascular disease: friend or foe.</a> Arterioscler Thromb Vasc Biol. 31 (1), 4-9 (2011).</li><li>Martinez, M. C., Tual-Chalot, S., Leonetti, D., Andriantsitohaina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+targets+and+tools+in+cardiovascular+disease.">Microparticles: targets and tools in cardiovascular disease.</a> Trends Pharmacol Sci. 32 (11), 659-665 (2011).</li><li>Benameur, T., Andriantsitohaina, R., Martinez, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+plasma+membrane-derived+microparticles.">Therapeutic potential of plasma membrane-derived microparticles.</a> Pharmacol Rep. 61 (1), 49-57 (2009).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocytic+microparticles+inhibit+angiogenesis+by+stimulating+oxidative+stress+and+negatively+regulating+VEGF-induced+pathways.">Lymphocytic microparticles inhibit angiogenesis by stimulating oxidative stress and negatively regulating VEGF-induced pathways.</a> Am J Physiol Regul Integr Comp Physiol. 294 (2), 467-476 (2008).</li><li>Yang, C., Gagnon, C., Hou, X., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+density+lipoprotein+receptor+mediates+anti-VEGF+effect+of+lymphocyte+T-derived+microparticles+in+Lewis+lung+carcinoma+cells.">Low density lipoprotein receptor mediates anti-VEGF effect of lymphocyte T-derived microparticles in Lewis lung carcinoma cells.</a> Cancer Biol Ther. 10 (5), 448-456 (2010).</li><li>Angelillo-Scherrer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-derived+microparticles+in+vascular+homeostasis.">Leukocyte-derived microparticles in vascular homeostasis.</a> Circ Res. 110 (2), 356-369 (2012).</li><li>Maeno, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+Cells+are+required+for+inflammation+and+destruction+in+cigarette+smoke-induced+emphysema+in+mice.">CD8+ T Cells are required for inflammation and destruction in cigarette smoke-induced emphysema in mice.</a> J Immunol. 178 (12), 8090-8096 (2007).</li><li>Qiu, Q., Xiong, W., Yang, C., Gagnon, C., Hardy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte-derived+microparticles+induce+bronchial+epithelial+cells'+pro-inflammatory+cytokine+production+and+apoptosis.">Lymphocyte-derived microparticles induce bronchial epithelial cells' pro-inflammatory cytokine production and apoptosis.</a> Mol Immunol. 55 (3-4), 220-230 (2013).</li><li>Martin, 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=Shed+membrane+particles+from+T+lymphocytes+impair+endothelial+function+and+regulate+endothelial+protein+expression.">Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.</a> Circulation. 109 (13), 1653-1659 (2004).</li><li>Shet, A. 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=Sickle+blood+contains+tissue+factor-positive+microparticles+derived+from+endothelial+cells+and+monocytes.">Sickle blood contains tissue factor-positive microparticles derived from endothelial cells and monocytes.</a> Blood. 102 (7), 2678-2683 (2003).</li><li>Mause, S. F., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles:+protagonists+of+a+novel+communication+network+for+intercellular+information+exchange.">Microparticles: protagonists of a novel communication network for intercellular information exchange.</a> Circ Res. 107 (9), 1047-1057 (2010).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-proliferative+and+anti-tumour+effects+of+lymphocyte-derived+microparticles+are+neither+species-+nor+tumour-type+specific.">Anti-proliferative and anti-tumour effects of lymphocyte-derived microparticles are neither species- nor tumour-type specific.</a> J Extracell Vesicles. 3, (2014).</li><li>Soleti, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticles+harboring+Sonic+Hedgehog+promote+angiogenesis+through+the+upregulation+of+adhesion+proteins+and+proangiogenic+factors.">Microparticles harboring Sonic Hedgehog promote angiogenesis through the upregulation of adhesion proteins and proangiogenic factors.</a> Carcinogenesis. 30 (4), 580-588 (2009).</li></ol>

חברי פרלמנט הם מתווכים פעילים דיבורים צלב אינטר והמחקר שלהם הוא מבטיח בתחומים רבים של מדע. 11 מחקר זה הוצג פרוטוקול מפורט לדור בקנה מידה גדול במבחנה של LMPs נגזר משורת תאי T אפופטוטיים. חברי פרלמנט אלה מבטאים רפרטואר גדול של מולקולות הלימפוציטים והם מעורבים מ?...

Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane&#8211;derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation. 
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.

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			<td height="21" style="height:21px;width:303px;">LMPs production and characterization</td>
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generation of lymphocytic microparticles and detection of their proapoptotic effect on airway epithelial cells

העניין בתפקידים הביולוגיים של שלפוחית-נגזר קרום תא בתקשורת תאי תאים גדל בשנים האחרונות. Microparticles (חברי פרלמנט) הוא סוג אחד כזה של שלפוחית, הנע בקוטר מ 0.1 מיקרומטר עד 1 מיקרומטר, ולשפוך בדרך כלל מקרום הפלזמה של תאים האיקריוטים עוברים הפעלה או אפופטוזיס. כאן אנו מתארים את הדור של microparticles-נגזרים הלימפוציטים T (LMPs) מתאי T CEM אפופטוטיים מגורה עם actinomycin LMPs ד מבודד באמצעות תהליך רב שלבי צנטריפוגה ההפרש ומאופיין באמצעות cytometry זרימה. פרוטוקול זה גם מציג שיטת זיהוי מוות של תאים באתר עבור הוכחת השפעת proapoptotic של LMPs על תאי האפיתל הסימפונות נגזרים מexplants העכבר עיקרי הנשימה הסימפונות רקמה. שיטות שתוארו במסמך זה מספקים הליך שחזור לבידוד כמויות שופעות של LMPs לימפוציטים אפופטוטיים במבחנה. LMPs נגזרבאופן זה ניתן להשתמש כדי להעריך את המאפיינים של דגמי מחלה שונים, ולפרמקולוגיה וטוקסיקולוגיה בדיקות. בהתחשב בכך שאפיתל דרכי הנשימה מציע מכשול פיזי ופונקציונלי הגנה בין הסביבה החיצונית והרקמה בסיסית, שימוש בexplants רקמת הסימפונות ולא שורות תאי אפיתל הונצחו מספק מודל אפקטיבי לחקירות הדורשות רקמות בדרכי נשימה.

LMPs אופיינו עם Annexin V צביעה 10 על ידי תא הקרינה המופעל מיון ניתוח (FACS) ונשלטים באמצעות 1 מיקרומטר חרוזים שבי 97% מחברי הפרלמנט (≤1 מיקרומטר) היו חיובי Annexin-V-Cy5 (איור 1 א ו -1 B). בדרך כלל, כ -2.5 מ&quot;ג של LMPs התקבל בעקבות פרוטוקול זה. explants רקמת הסימפונות מעכברי C57BL / 6 הי?...

Watch this Scientific Journal Video about Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells at JoVE.com

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells (Video) | JoVE

microparticles Cell-לשפוך קרום (חברי פרלמנט) הוא שלפוחית ​​ביולוגית פעילה שיכול להיות מבודדת ואפקטי pathophysiological נחקרו בדגמים שונים. כאן אנו מתארים שיטה ליצירת חברי פרלמנט הנגזרים מלימפוציטים מסוג T (LMPs) ועבור הוכחת השפעת proapoptotic בתאי האפיתל בדרכי נשימה.

דור של Lymphocytic microparticles וזיהוי של Proapoptotic השפיעו על תאי אפיתל Airway

Interest in the biological roles of cell membrane&#8211;derived vesicles in cell&#8211;cell communication has increased in recent years. Microparticles ...

Research

JoVE Journal

Immunology and Infection

Generation of Lymphocytic Microparticles and Detection of their Proapoptotic Effect on Airway Epithelial Cells

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הקמת תרבות הנוזלית מכוסית של Airway האנושי המקוטב-3 Calu תאי אפיתל ללמוד תגובת תא מארחת לפתוגנים נשימה<em&gt; במבחנה</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

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Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver ...

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טיהור בקנה מידה גדולה של מגזרי בשר חזירים או שור קולטי אור חיצוני למבחני phagocytosis על תאי הרשתית פיגמנט אפיתל

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

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Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

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לא פולשנית דגימה של נוזל שורות Mucosal עבור כימות של In Vivo דרכי הנשימה העליונות החיסון-מגשרת רמות

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Polymorphonuclear neutrophil-derived microparticles (PMN)-MPs) are lipid bilayer, spherical microvesicles with sizes ranging from 50&#8211;1,000 nm in ...