Name: methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3d
Uploaded: 2017-03-16T13:00:00
Description: Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

We thank Mr. Christopher Manzella, MD/PhD student in the RKG laboratory, for helping to edit and proofread our manuscript.

1. Undersøgelse af Intestinal Transporters i en 3D Kultur System af Caco-2: Dyrkning 3D Caco-2 Cell Culture på en Gelagtig Protein Blanding <ol><li> Tø vækstfaktor reduceret gelatinøse proteinblanding på is i 8 timer (eller O / N) ved 4 ° C. Når optøet, gør 1 ml eller 500 pi prøver til brug eller opbevares ved -20 ° C til senere brug. </li><li> På dagen for kultur, forkøling dyrkningspladerne (for RNA og protein-ekstraktion) eller kamre objektglas (til immunfarvning) på is. Tilsæt 30 pi gelatinøse pro...

<ol>
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Fuldt differentierede Caco-2 cellemonolag stor udstrækning blevet anvendt som polariseret epiteliale monolag at studere intestinal transport 10, 11, 12, 13, 14, 15. Men for at efterligne den fysiologiske tilrettelæggelse af intestinale epitelceller, har der været betydelig interesse i udviklingen af ​​en Caco-2 kultur...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.
One of the authors&#39; names was corrected from:
Arivarasu N. Anabazhagan
to:
Arivarasu N. Anbazhagan

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.

Erratum: Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Tarmepitelet er udstyret med forskellige transportproteiner (kanaler, ATPaser, co-transportører og varmevekslere), der udfører mange funktioner lige fra absorptionen af ​​næringsstoffer, elektrolytter og lægemidler til sekretion af væske og ioner i lumen. Transportproteiner generere elektrokemiske gradienter, der tillader bevægelse af ioner eller molekyler i en vektorielt måde. Dette opnås ved de asymmetriske fordelinger af transportsystemerne i de apikale og basolaterale membraner af polariserede epitelceller. Desuden tight junctions, som tøjr tilstødende epitelceller, spiller en vigtig rolle i denne proces ved at tjene som en barriere for intramembrane diffusion af komponenter mellem de apikale og basolaterale membran domæner. Passende modelsystemer efterligner disse karakteristiske træk ved den indfødte tarmen (dvs. polaritet, differentiering og tight junction integritet) er afgørende for studiet af functionalitet af epitel transportsystemer. Med hensyn til modeller, de typiske cellelinjer anvendes i øjeblikket i intestinal epitel transportforskning er Caco-2, en model af fuldt differentieret, absorberende små intestinale epitelceller; og T84 celler eller HT-29 subkloner, modeller af krypt-afledte store tarmepitelceller 1. Konventionelt er disse cellelinjer dyrket som monolag i plastoverflader eller coatede transwell inserts. Trans cellekulturplade skær til en vis grad ligner in vivo-miljø ved at lade polariserede celler at fodre basolateralt. en begrænsning i konventionelle 2D dyrkningssystemer er imidlertid, at cellerne er tvunget til at tilpasse sig en kunstig, flad og stiv overflade. Således er den fysiologiske kompleksitet native epitel ikke er korrekt gengivet i en 2D-system. Denne begrænsning er blevet overvundet ved fremgangsmåder at dyrke celler i 3D i et specifikt mikromiljø, såsom gelatinøse protein mixture, som indeholder en række ekstracellulære matrixkomponenter 2, 3. Ikke desto mindre kan de in vitro-kulturerne ikke simulere kompleksiteten af tarmepitelet, som har flere celletyper og områdespecifik arkitektur i tarmen. Således undersøgelser med anvendelse af cellekultur modeller kræver yderligere validering i native tarmen. Anvendelse af in vitro 3D cellekultur og mus tarmslimhinden beskriver vi her enkle metoder til at studere reguleringen af den intestinale serotonin transporter (SLC6A4, SERT). Den SERT transportør regulerer ekstracellulære tilgængelighed af et vigtigt hormon og neurotransmitter, 5-hydroxytryptamin (5-HT), ved hurtigt at transportere den gennem en Na + / Cl - -afhængig proces. SERT er en kendt mål for anti-depressiva og har for nylig vist sig som en hidtil ukendt terapeutisk mål for GI lidelser, såsom diarré og tarmbetændelse.Metoder til at undersøge 5-HT-optagelse i intestinale epitelceller er tidligere blevet beskrevet. For eksempel er Caco-2 celler dyrket på plast understøtninger eller permeable indsatse vist sig at udvise fluoxetin-sensitive 3H-5-HT-optagelse i både apikale og basolaterale domæner 4. Målingen af SERT funktion i isolerede Brush membranvesikler (BBMVs) fremstillet fra humant organdonorens tyndtarmen er også blevet beskrevet af US 5. 3H-5-HT-optagelse i humane BBVMs blev vist at være fluoxetin-følsomme og Na + / Cl - -afhængig, og det udviste mætningskinetik med en Km på 300 nM 5. Ved hjælp af lignende fremgangsmåder vi også tidligere målt SERT funktion som 3H-5-HT-optagelse i muse intestinale BBMVs 6. Men fremstillingen af ​​rene plasma membranvesikler kræver en stor mængde væv slimhinde,. Andre fremgangsmåder, såsom radiographic visualisering af 3H-5-HT optagelsessteder, er også tidligere blevet vist i marsvin og rotte tyndtarmen 7. Ussing kammer giver en mere fysiologisk system til at måle transport af ioner, næringsstoffer og lægemidler i forskellige epitelvæv. Den største fordel ved Ussing kammer teknik er, at den muliggør præcis måling af de elektriske og transport parametre for intakt, polariseret tarmepitelet. Endvidere at mindske virkningen af den indre neuromuskulære system, kan udføres seromuscular stripning af tarmslimhinden til at undersøge reguleringen af transportører i epitelet 8. Vi viser, at Caco-2 celler dyrket i 3D på gelatinøse protein udgøre en hul lumen udtrykker forskellige apikale og basolaterale markører. Disse celler udviser højere ekspression af SERT end 2D Caco-2-celler. Metoder til at vokse 3D-celler og til PErform immunfarvning eller RNA og protein-ekstraktion er beskrevet. Derudover beskriver vi metoder til at studere SERT funktion og regulering af TGF-β1, en pleiotropisk cytokin i tyndtarmens slimhinde ved at udnytte en Ussing kammer teknik.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>10X PBS</td>
			<td>ATCC</td>
			<td>ATCC&#174; HTB&#173;37&#8482;</td>
			<td>Cells</td>
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		<tr>
			<td>10X PBS</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Microsope for confocal Imaging</td>
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			<td>3[H]-5-HT&#160;</td>
			<td>LabtekII</td>
			<td>152453</td>
			<td>Culturing cells for microscopy</td>
		</tr>
		<tr>
			<td>5-HT&#160;</td>
			<td>Gibco</td>
			<td>70013-032</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>95%O2- 5%Co2 cylinder</td>
			<td>KPL</td>
			<td>71-00-27</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Agar (for Agar plates)</td>
			<td>Fischer</td>
			<td>BP151-100</td>
			<td>Acute toxicity, Ora</td>
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		<tr>
			<td>Agar (for electrode)</td>
			<td>Sigma</td>
			<td>G7126-1KG</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Alexa FluorPhalloidin</td>
			<td>Sigma</td>
			<td>C3867-1VL</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>P6148-500G</td>
			<td>Harmful if swallowed or if inhaled</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>LifeTechnologies</td>
			<td>A12380</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>CaCo2 Cells</td>
			<td>Sigma</td>
			<td>A8806-5G</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Cell lysis Buffer&#160;</td>
			<td>Fischer</td>
			<td>BP337-100</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Chabered slides</td>
			<td>Sigma</td>
			<td>S5886-1KG</td>
			<td>Not a hazardous substance</td>
		</tr>
		<tr>
			<td>Collagen Type-1Solution</td>
			<td>Sigma</td>
			<td>S8045-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrodes</td>
			<td>Sigma</td>
			<td>S-0876</td>
			<td></td>
		</tr>
		<tr>
			<td>EMEM</td>
			<td>Gibco by Life Technologies</td>
			<td>25200-056</td>
			<td></td>
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		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Used as Extracellular matrix</td>
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			<td>Fluoxetine</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
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			<td>&#160;Gentamicin</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>Cell culture Media</td>
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			<td>Glycin</td>
			<td>Cell Signaling, Danvers, MA</td>
			<td></td>
			<td></td>
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			<td>Hepes&#160;</td>
			<td>Roche</td>
			<td>11836145001</td>
			<td></td>
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			<td>Indomethacin</td>
			<td>Gibco by Life Technologies</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Gibco by Life Technologies</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Gibco by Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
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			<td>L-ascorbic acid&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>10082147</td>
			<td></td>
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			<td>Liquid scintillation counter&#160;</td>
			<td>Gibco by Life Technologies</td>
			<td>70013--032</td>
			<td></td>
		</tr>
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			<td>LSM710 Meta</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>EM-CSYS-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2304</td>
			<td></td>
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		<tr>
			<td>Matrigel</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>VCC MC8</td>
			<td></td>
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			<td>MgCl2</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>DM MC6</td>
			<td></td>
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		<tr>
			<td>Multichannel Voltage/current Clamp</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2300</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Physiologic Instruments, San Diego, CA&#160;</td>
			<td>P2023-100</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (10%)</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>S233</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>P288</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strp</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein assay reagent</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>10420</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase Inhibitor Cocktail</td>
			<td>MEDOX, Chicago, IL&#160;</td>
			<td>style G- 12300</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA easy Mini kit</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel Electrode Input Module&#160;</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>A92902</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliders for snapwell Chambers</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>H9523</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Na2HPO4)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>F132</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-Hydroxide (NaOH)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>T7039</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;1</td>
			<td>Perkin-Elmer,Waltham, MA</td>
			<td>NFT1167250UC</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Packard Instruments, Downers Grove, IL</td>
			<td>B1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Bio Rad Laboratories, Hercules, CA</td>
			<td>5000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Ussing Chamber (chamber only)</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ussing Chamber Systems</td>
			<td>Sigma-Aldrich, St Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods to study epithelial transport protein function and expression in native intestine and caco-2 cells grown in 3d

Tarmepitelet har vigtige transport- og barriere funktioner, der spiller afgørende roller i normale fysiologiske funktioner i kroppen, samtidig med at en barriere for fremmede partikler. Nedsat epitelial transport (ion, næringsstof eller narkotika) er blevet forbundet med mange sygdomme og kan have konsekvenser, der rækker ud over de normale fysiologiske funktioner på transportørerne, såsom ved at påvirke epitelial integritet og tarmen microbiome. Forståelse af funktion og regulering af transportproteiner er afgørende for udvikling af forbedrede terapeutiske interventioner. Den største udfordring i studiet af epitelial transport er ved at udvikle en egnet model, der sammenfatter vigtige elementer i de indfødte intestinale epitelceller. Adskillige in vitro-cellekultur modeller, såsom Caco-2, T-84, og HT-29-Cl.19A celler anvendes typisk i epitel transportforskning. Disse cellelinier repræsenterer en reduktionistisk tilgang til modellering af epithelium og har været brugt i mange mekanistiske undersøgelser, herunder deres undersøgelse af epitel-mikrobielle interaktioner. Men cellemonolag afspejler ikke celle-celle-interaktioner og in vivo mikromiljø. Celler dyrket i 3D har vist sig at være lovende modeller for permeabilitet lægemiddel undersøgelser. Vi viser, at Caco-2-celler i 3D kan bruges til at studere epiteliale transportører. Det er også vigtigt, at undersøgelser i Caco-2 celler er suppleret med andre modeller at udelukke cellespecifikke virkninger og tage hensyn til kompleksiteten af ​​den native tarmen. Adskillige fremgangsmåder er tidligere blevet anvendt til at vurdere funktionaliteten af ​​transportører, såsom udkrængede sac og optagelse i isolerede epitelceller eller i isolerede plasma membranvesikler. Under hensyntagen til udfordringerne på området med hensyn til modeller og måling af transport-funktion, vi demonstrerer her en protokol til at vokse Caco-2 celler i 3D og beskrive brugen af ​​et Ussing kammer som eneffektiv fremgangsmåde til måling af serotonin transport, såsom i intakte polariserede intestinale epitel.

Immunofarvning i 3D Caco-2 cyster er vist i figur 1. Et repræsentativt billede af en XY-planet, der viser velafgrænsede lumen i 3D-cyste farvet med phalloidin actin er afbildet i figur 1A. Den side der vender lumen betegner den apikale side. Epitelceller dyrket i et 3D-miljø, resulterer i en robust epitel fænotype. Forskellige Caco-2 cyster på dag 12, når actin og SERT blev co-farvet, er vist i figur 1B. I denne gengivelse af en XY...

Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Vi beskriver enkle metoder til at studere reguleringen af intestinal serotonin transporter (SERT) funktion og ekspression anvendelse af en in vitro cellekultur-model af Caco-2-celler dyrkes i 3D og en ex vivo model for muse tarmene. Disse metoder kan anvendes til studiet af andre epitel transportører.

Metoder til at studere Epithelial Transport Protein Funktion og Expression i Native tarmen og Caco-2 celler dyrket i 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Research

JoVE Journal

Biology

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Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

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Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...