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 arbejde blev støttet af tilskud fra Fritz Thyssen Foundation (10.11.2.153 til RK), den tyske forskningsråd (DFG, KR2119/3-2 og KR2119/8-1 til RK), Forbundsministeriet for Uddannelse og Forskning (BMBF , NGFNplus; 01GS08134 til RK) og en ph.d.-stipendium fra den velgørende Hertie Foundation [til LFB]. Vi takker Carolin Obermaier og Julia Westermeier for deres støtte under videooptagelse.

1. Hudbiopsi og Dyrkning af humane fibroblaster <ol><li> Den hudbiopsi skal tages af en erfaren læge. Proceduren foregår under sterile forhold og kræver lokalbedøvelse. Typiske steder, der anvendes til biopsi er den indvendige side af den øverste arm, skulder eller nederste del af ryggen. </li><li> Du kan tage 4x4mm eller 6x6mm diameter prøve af punch biopsi for at få nok væv til dyrkning af humane fibroblaster. </li><li> Skær hudbiopsi i lige små stykker under sterile betingelser for at adskille dem i 2-4 T...

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Patient hudfibroblaster som ex vivo modeller repræsenterer et vigtigt redskab til at karakterisere sygdoms-associerede genetiske defekter. Desuden er hud-afledte fibroblaster let tilgængelig og kan udvides efter dyrkning. Derfor primære celler opnået fra patienter, der bærer PD-forbundne genetiske mutationer foretrækkes i forhold til anvendelsen af ​​tumorcellelinier, som de indeholder ikke kun det endogene sygdomsfremkaldende gen, men hele den genetiske baggrund af det angrebne individ. Desuden er de...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalog nr. </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, uden phenolrødt </td><td> Invitrogen </td><td> 11835-063 </td></tr><tr><td> Dulbeccos phosphatbufrede saltvand (DPBS) </td><td> Invitrogen </td><td> 14190-094 </td></tr><tr><td> Fibroblastvækstfaktor 2 (FGF2) </td><td> PeproTech </td><td> 100-18B </td></tr><tr><td> Accumax (løsrivelse opløsning) </td><td> PAA </td><td> L11-008 </td></tr><tr><td> Lab-TekTMII kamre dækglasset </td><td> Nalge Nunc International </td><td> 115.382 </td></tr><tr><td> Tetramethylrodamine ethylester (TMRE) </td><td> Invitrogen </td><td> T-669 </td></tr><tr><td> Mitotracker Green 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> Tabel 1. Specifikke reagenser og udstyr.

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

Parkinsons sygdom (PD) er den næsthyppigste bevægelse uorden og påvirker 1% af mennesker over en alder af 60 1. Fordi aldring er den vigtigste risikofaktor, vil tilfælde af PD stige i løbet af de næste årtier 2. Ved siden af patologisk proteinfoldning og værdiforringede protein nedbrydningsveje blev ændringer af mitokondriefunktionen og morfologi påpeget som yderligere kendetegnende for neurodegeneration i PD 3-11. Efter flere års forskning i murine og humane cancerceller som in vitro-modeller at dissekere molekylære veje for Parkinsonisme, er brugen af humane fibroblaster fra patienter og passende kontroller som ex vivo modeller blive et værdifuldt forskning værktøj, hvis potentielle forbehold tages i betragtning. Andre end udødeliggjorte, snarere kunstige cellemodeller, primære fibroblaster fra patienter, der transporterer sygdom-associerede mutationer tilsyneladende afspejler vigtige patologiske træk of den menneskelige sygdom. Her har vi afgrænse procedure for at tage hudbiopsier, dyrkning af humane fibroblaster og bruge detaljerede protokoller for væsentlige mikroskopiske teknikker til at definere mitokondriske fænotyper. Disse blev anvendt til at undersøge forskellige træk forbundet med PD som er relevante for mitokondrie-funktion og dynamik. Ex vivo, kan mitokondrier analyseres med hensyn til deres funktion, morfologi, colokalisering med lysosomer (organellerne nedbrydning dysfunktionel mitokondrier) og nedbrydning via lysosomal omsætningsvej . Disse fænotyper er særdeles relevante for identifikationen af ​​tidlige tegn på PD og kan gå forud kliniske motoriske symptomer i humane sygdom-genbærere. Derfor kan assayene præsenteret her anvendes som værdifulde værktøjer til identifikation patologiske træk ved neurodegeneration og bidrager til at 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 patienter med mutationer i Parkinsons sygdomsfremkaldende gener udgør en lettilgængelig<em&gt; Ex vivo</em&gt; Model til at studere sygdom-associerede fænotyper. Live-cell imaging giver mulighed for at studere morfologiske og funktionelle parametre i levende celler. Her beskrives fremstillingen af ​​humane fibroblaster og efterfølgende overvågning af mitokondrielle fænotyper.

Anvendelsen af ​​primære humane fibroblaster til overvågning Mitochondriale fænotyper i Field af Parkinsons sygdom

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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Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Anvendelsen af ​​primære humane fibroblaster til overvågning Mitochondriale fænotyper i Field af Parkinsons sygdom

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