Name: the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease
Uploaded: 2012-10-03T13:00:00
Description: Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

Dette arbeidet ble støttet med tilskudd fra Fritz Thyssen Foundation (10.11.2.153 til RK), den tyske Research Council (DFG, KR2119/3-2 og KR2119/8-1 til RK), den føderale departementet for utdanning og forskning (BMBF , NGFNplus; 01GS08134 til RK) og av en doktor stipend fra den veldedige Hertie Foundation [til LFB]. Vi takker Carolin Obermaier og Julia Westermeier for deres støtte under videoen skyte.

1. Hudbiopsi og Dyrking av humane fibroblaster <ol><li> Den hudbiopsi må tas av en erfaren lege. Prosedyren foregår under sterile betingelser og krever lokal anestesi. Typiske områder som brukes for biopsi er den indre side av den øvre arm, skulder eller korsryggen. </li><li> Du kan ta 4x4mm eller 6x6mm diameter prøven ved punch biopsi for å få nok vev for dyrking humane fibroblaster. </li><li> Skjær hudbiopsi i like små biter under sterile forhold for å skille dem i 2-4 T25 flasker. Hver kolbe bør innehold...

<ol>
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Pasient hudfibroblaster som ex vivo-modeller representerer et viktig verktøy for å karakterisere sykdomsassosierte genetiske defekter. I tillegg, hud-avledede fibroblaster er lett tilgjengelige og kan utvides ved dyrkning. Derfor, primære celler oppnådd fra pasienter som frakter PD-assosierte genetiske mutasjoner er å foretrekke fremfor bruk av tumorcellelinjer som de inneholder ikke bare den endogene sykdomsfremkallende genet, men hele genetiske bakgrunnen for berørte enkelte. Videre har de vist seg å f...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Selskapet </td><td> Katalog nei. </td></tr><tr><td> Roswell Park Memorial Institute (RPMI) 1640 medium </td><td> Invitrogen </td><td> 52400-025 </td></tr><tr><td> RPMI 1640-medium, ingen fenolrødt </td><td> Invitrogen </td><td> 11835-063 </td></tr><tr><td> Dulbeccos fosfatbufret saltløsning (DPBS) </td><td> Invitrogen </td><td> 14190-094 </td></tr><tr><td> Fibroblast vekstfaktor 2 (FGF2) </td><td> PeproTech </td><td> 100-18B </td></tr><tr><td> AccuMax (avløsning løsning) </td><td> PAA </td><td> L11-008 </td></tr><tr><td> Lab-TekTMII Chambered coverglasses </td><td> Nalge Nunc International </td><td> 115382 </td></tr><tr><td> Tetramethylrodamine etylester (TMRE) </td><td> Invitrogen </td><td> T-669 </td></tr><tr><td> Mitotracker Grønn FM </td><td> Invitrogen </td><td> M-7514 </td></tr><tr><td> Mitotracker CM-H2XRos </td><td> Invitrogen </td><td> M-7513 </td></tr><tr><td> Lyostracker Red DND-99 </td><td> Invitrogen </td><td> L-7528 </td></tr><tr><td> Hoechst 33342 </td><td> Invitrogen </td><td> H-3570 </td></tr></tbody></table> Tabell 1. Spesifikke reagenser og utstyr.

the use of primary human fibroblasts for monitoring mitochondrial phenotypes in the field of parkinson's disease

Parkinsons sykdom (PD) er den nest vanligste bevegelse lidelse og rammer 1% av personer over 60 år en. Fordi aldring er den viktigste risikofaktor, vil tilfeller av PD øke i løpet av de neste tiårene 2. Ved siden av patologisk protein folding og nedsatt protein degradering pathways, ble endringer i mitokondrienes funksjon og morfologi påpekt som ytterligere kjennetegn på neurodegeneration i PD 3-11. Etter års forskning i murine og humane kreftceller som in vitro modeller for å dissekere molekylære mekanismene i parkinsonisme, har bruken av menneskelige fibroblaster fra pasienter og hensiktsmessige kontroller som ex vivo-modeller blitt et verdifullt verktøy for forskning, hvis potensielle begrensningene er vurdert. Annet enn udødeliggjort, heller kunstige celle-modeller, primære fibroblaster fra pasienter bærer sykdom mutasjoner tilsynelatende reflekterer viktige patologiske funksjoner of sykdom hos mennesker. Her har vi avgrense prosedyren for å ta hud biopsier, dyrking humane fibroblaster og ved hjelp av detaljerte protokoller for viktige mikroskopiske teknikker for å definere mitokondrie fenotyper. Disse ble brukt til å undersøke ulike funksjoner knyttet til PD som er relevante for mitokondrie funksjon og dynamikk. Ex vivo, kan mitokondrier bli analysert i forhold til sin funksjon, morfologi, colocalization med lysosomer (organelles nedverdigende dysfunksjonelle mitokondrier) og nedbrytning via lysosomal vei . Disse fenotyper er svært relevant for identifisering av tidlige tegn på PD og kan gå forut kliniske motoriske symptomer i menneskelig sykdom-genet. Derfor kan analysene som presenteres her brukes som verdifulle verktøy for å identifisere patologiske trekk ved neurodegeneration og bidra til å definere nye terapeutiske strategier i PD.

Watch this Scientific Journal Video about The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease at JoVE.com

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Fibroblaster fra pasienter bærer mutasjoner i Parkinsons sykdom-forårsaker gener representerer en lett tilgjengelig<em&gt; Ex vivo</em&gt; Modell for å studere sykdomsassosierte fenotyper. Levende celle bildebehandling gir mulighet til å studere morfologiske og funksjonelle parametre i levende celler. Her beskriver vi utarbeidelse av humane fibroblaster og påfølgende oppfølging av mitokondrie fenotyper.

Bruk av primære humane fibroblaster for overvåking Mitokondrielle Fenotyper i feltet av Parkinsons sykdom

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

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