Name: generation of genetically modified organotypic skin cultures using devitalized human dermis
Uploaded: 2015-12-14T14:00:00
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Dette arbeidet var supportedby American Cancer Society stipendiater Grant (RSG-12-148-01-DDC) og CIRM Basic Biology Award (RB4-05779).

Den menneskelige huden protokollen ble utført i samsvar med retningslinjene fra University of California, San Diego forskningsetiske komité. Menneskelig hud kan fås fra kasserte kirurgiske prøver eller kjøpt fra hud banker (hud bank er oppført i tabellen materiell / utstyr). Stedet hvor huden er avledet fra eller alder av donor er ikke kritisk for forsøket så lenge basalmembransonen proteiner (kollagen / laminin) i dermis ikke er degradert. 1. Utarbeid...

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Genetisk manipulasjon i human hud organotypiske kulturer tilbyr mange fordeler på vanlige studert 2D dyrkede celler samt musemodeller. 2D kulturer mangler de tredimensjonale celle-celle og celle-ekstracellulær matriks-interaksjoner er funnet i intakt vev og organer. Nyere studier har også funnet store forskjeller mellom 2D- og 3D-dyrkede hud kreftceller med 3D-dyrkede celler som viser mye mer genuttrykk likheter til primær menneskelig hud svulster 16. Humane organotypiske hud kulturer har også flere ford...

De menneskelige epidermis er et stratifisert epitel som kobles til de underliggende dermis gjennom en ekstracellulær matrise kjent som den basalmembran zone.The epidermis ikke bare tjener som en ugjennomtrengelig barriere for å hindre tap av fuktighet, men også som en første forsvarslinje for å beskytte kroppen fra utenlandske og giftige stoffer 1. Basallaget, som er det dypeste lag av epidermis, inneholder epidermal stamceller og forløperceller som gir opphav til den differensierte avkom som danner resten av epidermis 2. Som epidermale progenitorceller differensiere de vandrer oppover for å danne det første sjikt av differensierte celler som kalles spinous laget 3. I spinous lag, celler slå på ekspresjon av keratin 1 og 10, som deretter gir styrke til å motstå fysisk stress for de differensierte lag av epidermis. Som spinous lagscellene ytterligere differensiere, de beveger seg oppover i epidermis til form den granulære lag som er karakterisert ved dannelse av keratohyalin og lamellære granuler, så vel som strukturelle proteiner som er montert under plasmamembranen. Ettersom cellene gå frem på denne differensieringsprosessen proteinene under plasmamembranen er kryss knyttet til hverandre mens de lamellære granuler ekstrudert fra cellene for å danne en lipid rik barriere kalt stratum corneum 4. Sykdommer som involverer endringer i epidermal vekst og differensiering innvirkning ~ 20% av befolkningen 5. Således forstå mekanismene for denne fremgangsmåte er av stor betydning. Siden manifestasjon av mange av disse sykdommene er betinget av celle-celle eller celle-matriks kontakt, har organotypiske kulturer hvor human epidermis blir rekonstituert i et 3D-miljø er skapt 6-10. Disse fremgangsmåtene involverer typisk bruken av primære eller transform keratinocytter sådd på ekstracellulær matriks som devitalized humandermis, Matrigel eller kollagen. For å forstå gen regulerende mekanismer som er viktige i epidermal vekst og differensiering, kan keratinocytter være genetisk manipulert gjennom retrovirale vektorer til knockdown eller overuttrykke gener i 2D kultur og deretter rekonstituert i 3D. Disse metodene har vært brukt mye til å karakterisere gener involvert i epidermal stilk og stamcelleselvfornyelse og differensiering samt progresjon til neoplasi 11-21. Her er en grundig protokollen på hvordan å endre genuttrykk i epidermal organotypiske kulturer gjennom bruk av retrovirus gitt.

<table>
	<tbody>
		<tr>
			<td>Human skin</td>
			<td>New York Firefighters Skin Bank</td>
			<td><a href="http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html">http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html</a></td>
		</tr>
		<tr>
			<td>PEN/STREP</td>
			<td>GIBCO</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>amphotropic phoenix cell lines</td>
			<td>ATCC</td>
			<td>CRL-3213</td>
		</tr>
		<tr>
			<td>FUGENE 6 transfection reagent</td>
			<td>Promega</td>
			<td>E2691</td>
		</tr>
		<tr>
			<td>Keratinocyte Media (KCSFM)</td>
			<td>Life Technologies</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>11995</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Cambrex</td>
			<td>12-615F</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>10437-028</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A-9795</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma</td>
			<td>&#160;C-8052</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Calbiochem</td>
			<td>3896</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I-1882</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Invitrogen</td>
			<td>13247-051</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma</td>
			<td>T-0665</td>
		</tr>
		<tr>
			<td>Ciprofloxacin Hydrochloride</td>
			<td>Serologicals</td>
			<td>89-001-1</td>
		</tr>
		<tr>
			<td>cautery</td>
			<td>Bovie Medical Corporation</td>
			<td>AA01</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
		</tr>
		<tr>
			<td>Keratin 1 antibody</td>
			<td>Biolegend</td>
			<td>PRB-149P</td>
		</tr>
		<tr>
			<td>square pegs</td>
			<td>Arts and crafts stores</td>
			<td></td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>ATCC</td>
			<td>PCS-200-010</td>
		</tr>
		<tr>
			<td>human neonatal keratinocytes</td>
			<td>Cell Applications</td>
			<td>102K-05n</td>
		</tr>
		<tr>
			<td>MSCV retroviral vector</td>
			<td>Clontech</td>
			<td>634401</td>
		</tr>
		<tr>
			<td>LZRS retroviral vector</td>
			<td>Addgene</td>
			<td></td>
		</tr>
		<tr>
			<td>pSuper.Retro.Puro Retroviral vector</td>
			<td>Oligoengine</td>
			<td>VEC-PRT-0002&#160;</td>
		</tr>
		<tr>
			<td>hexadimethrine bromide&#160;</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
		</tr>
	</tbody>
</table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Organotypiske kulturer tillate omdanning av et 3D-miljø kritisk for celle-celle-kontakt og celle-matriks-interaksjoner som etterligner den fysiologiske funksjon og deres in vivo vev motstykker. Dette er eksemplifisert ved organotypiske hud kulturer som trofast rekapitulerer epidermal differensiering og stratifisering program. Primære human epidermal keratinocytter er genetisk manipulable gjennom retrovirus hvor gener kan lett overuttrykt eller slått ned. Disse genetisk modifiserte keratinocytter kan deretter brukes til å regenerere human epidermis i organotypiske hud kulturer som gir en kraftig modell for å studere genetisk trasé slag epidermal vekst, differensiering og utvikling av sykdommen. Protokollene som er presentert her for å beskrive fremgangsmåter for å fremstille devitalized menneskehuden, så vel som for å genetisk manipulere primære humane keratinocytter for å generere organotypiske hud kulturer. Regenereres menneskelig hud kan brukes i downstrEAM applikasjoner som genekspresjon profilering, farging, og kromatin immunoutfellinger etterfulgt av høy gjennomstrømming sekvensering. Således vil dannelse av slike genetisk modifiserte organotypiske hud kulturer tillate bestemmelse av gener som er kritisk for å opprettholde homeostase hud.

Det første trinnet i å generere organotypiske human hud er å fjerne epidermis fra dermis. De to ukers inkubering av huden ved 37 ° C i 4 x pen / strep / PBS bør tillate separasjon av dermis fra epidermis (figur 1A). Hvis skille epidermis og dermis er vanskelig deretter plassere vevet ved 37 ° C i 4x penn / strep / PBS for en uke, og deretter prøve peeling igjen med tang. En av nøklene til regenerering av human epidermis på devitalized dermis er å sikre at keratinoc...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Generering av genmodifiserte organotypiske Skin kulturer Bruke devitalized Menneskelig Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

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A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

Et modifisert Trier sosialteststest for sårbare meksikanske amerikanske ungdommer

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Generering av genetisk modifiserte mus gjennom mikroinjeksjon av oocytter

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

Generasjon og dyrking av primære menneskelige keratinocytter fra voksen hud

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

Tredimensjonale Organotypic kulturer av Vestibular og auditiv Sanseorganer

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...