Name: induction of an inflammatory response in primary hepatocyte cultures from mice
Uploaded: 2017-03-10T15:00:00
Description: Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Dette arbeidet ble støttet av NIH (R01HL128714 til CF) og (F31DK10236101 til KS) og American Heart Association (CF og AG).

Alle dyr protokoller og eksperimentelle prosedyrer ble godkjent av Institutional Animal Care og bruk Committee (IACUC) ved University of Miami Miller School of Medicine. 1. Forberedelse <ol><li> Forvarm lever perfusjon media og leveren fordøye media i et vannbad ved 37 ° C. </li><li> Sett opp en perfusjon pumpe (30 ml / min). forfyll nøye rørsystemet av pumpen med leveren perfusjon media. Unngå luftbobler i systemet og forberede stereomikroskop. </li><li> Coat cellekultur retter med k...

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Isolere primære hepatocytter fra mus er en rask, billig og pålitelig verktøy for å studere inflammatoriske responser ex vivo. Hvis utført riktig, kan resultatet lett bli generert og gjengitt i en tidsriktig og kostnadseffektiv måte. Følgende punkter bør vurderes nøye for å sikre en vellykket isolasjon. Den kirurgiske innsnitt og kanylering av IVC skal utføres under narkose og ikke etter aktiv dødshjelp. En ung, uerfaren etterforsker vil trenge mer tid i begynnelsen for ?...

Kronisk nyresykdom (CKD) kan defineres som en tilstand av akutt og kronisk inflammasjon 1. Hos pasienter med CKD, serumnivåer av den phosphaturic hormonet fibroblast vekstfaktor 23 (FGF23) progressivt stige for å opprettholde serumfosfat homeostase 2. Økte serum FGF23 nivåer er uavhengig assosiert med kardiovaskulær sykelighet og dødelighet blant pasienter som begynner hemodialyse behandling tre, fire. Videre har flere kliniske studier har vist en sterk korrelasjon mellom forhøyede nivåer FGF23 og serumnivåer av c-reaktivt protein (CRP), Interleukin-6 (IL-6) og tumornekrosefaktor α (TNFa) 5, 6. Videre, i en eksperimentell studie, vi har nylig vist at FGF23 kan direkte rettet mot hepatocytter og forårsake en betennelsesreaksjon ved å øke CRP og IL-6 production i leveren 7. Derfor, FGF23 kan fungere som en sirkulerende faktor som bidrar til systemisk inflammasjon i CKD. På begynnelsen av 70-tallet, primære hepatocytter ble isolert og undersøkt for første gang 8. Siden den primære dyrkede leverceller har blitt mye brukt for å undersøke metabolsk behandling, hormonal funksjon, legemiddelmetabolisme og giftighet, så vel som immunresponser og inflammatoriske responser 9, 10. Tidligere protokoller har i hovedsak beskrevet enzymatisk isolering av primære hepatocytter fra menneske levervev 11, 12. Mens en utmerket modell, etterlater denne ut muligheten til å studere hvordan genetisk manipulasjon påvirker komplekse leversignalmekanismer samt funksjonelle konsekvenser ved ulike typer stimuli. I det følgende vil vi beskrive isolering av murine primære hepatocytter. Spesielt kan virkningen av flere mediatorer av den hepatiske akutt fase respons, slik som lipopolysakkarid (LPS), IL-6 og FGF23 analyseres i en enkel, hurtig og reproduserbar måte 13. Heri presenterer vi en protokoll for den enzymatiske isolering av hepatocytter fra voksne mus, og vi vise at etablerte indusere av inflammasjon, slik som LPS og IL-6, så vel som nye inflammatoriske mediatorer slik som FGF23, kan direkte stimulere ekspresjon og sekresjon av inflammatoriske cytokiner, slik som CRP og IL-6 i dyrkede hepatocytter.

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induction of an inflammatory response in primary hepatocyte cultures from mice

Leveren spiller en avgjørende rolle i reguleringen av systemisk inflammasjon. Ved kronisk nyresykdom i særdeleshet, reagerer leveren i respons til uremisk miljø, oksidativt stress, endotoksemi og redusert clearance av sirkulerende proinflammatoriske cytokiner ved å fremstille et stort antall av akutt-fase reaktanter. Eksperimentelle verktøy for å studere betennelse og den underliggende rolle av hepatocytter er avgjørende for å forstå regulering og bidraget av lever cytokiner til en systemisk akutt faserespons og en forlenget pro-inflammatorisk banen, særlig i en innviklet omgivelser slik som kronisk nyresykdom. Etter å ha studert komplekse mekanismer for betennelse in vivo er fortsatt utfordrende, ressurskrevende og krever vanligvis bruk av transgene dyr, primære isolerte hepatocytter gi et robust verktøy for å få mekanistiske innsikt i lever akutt fase respons. Siden denne in vitro teknikk har moderate kostnader, høy reproducibility og felles teknisk kunnskap, primære isolerte hepatocytter kan også enkelt brukes som en screening tilnærming. Her beskriver vi en enzymatisk-basert metode for å isolere primære muse hepatocytter, og vi beskriver vurderingen av en betennelsesreaksjon i disse cellene ved hjelp av ELISA og kvantitativ real-time PCR.

histologi Representative lys mikroskopi bilder av primære isolerte og dyrkede celler er vist i figur 1A. Immunocytokjemisk analyse viser at isolerte hepatocytter sterkt uttrykker albumin (rød), så vel som fibroblast-vekstfaktor-reseptor 4 (FGFR4) (grønn). Kjerner er farget med 4 &#39;, 6-diamidino-2-fenylindol (DAPI) (blå). (Figur 1B). <p class="jove_step" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice at JoVE.com

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

Her viser vi en enzymatisk metode for å isolere primære hepatocytter fra voksne mus, og vi beskriver kvantifisering av en betennelsesreaksjon ved hjelp av ELISA og real-time PCR.

Induksjon av en betennelsesreaksjon i Primær hepatocytter kulturer fra Mus

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