Name: molecular imaging to target transplanted muscle progenitor cells
Uploaded: 2013-03-27T08:54:00
Description: Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Forfatterne vil gerne takke Sanjiv Gambhir for den gave af fluc/mrfp/sr39tk reportergenet. Dette arbejde blev finansieret af The Stem Cell Network (SCN) i Canada, og The Jesse Journey Foundation.

1. Opretholdelse og formering af C2C12 myoblaster <ol><li> Plade C2C12 myoblaster i en 75 cm2 kolbe og opretholde celler i højglucose Dulbeccos modificerede Eagles Serum (HG-DMEM) suppleret med føtalt bovint serum (FBS) til en slutkoncentration på 10%. Lad ikke celler til at blive sammenflydende til enhver tid, da dette vil nedbryder myoblastic befolkning. Medium ændres hver anden dag Note:. Altid varmt medium til 37 ° C i et vandbad inden anvendelse. </li><li> Når myoblaster bliver o...

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I denne undersøgelse har vi beskrevet en hurtig og pålidelig molekylær billeddannelse, reportergen tilgang til ikke-invasivt target myoblaster / Middelhavspartnerlandene efter transplantation. Selv om denne undersøgelse viser den korte lokalisering af transplanterede Middelhavspartnerlandene via bioluminescense imaging (BLI), den måde, hvorpå celler målrettes kan faktisk let anvendes til en langsgående vurdering af celle-transplantation, ved implantation af celler, der stabilt udtrykker reporte...

<table border="1"><tbody><tr><td> Navn på reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> C2C12 myoblaster </td><td> ATCC </td><td></td><td></td></tr><tr><td> Dulbeccos modificerede Eagles medium </td><td> Life Technologies </td><td> 12800-017 </td><td></td></tr><tr><td> føtalt bovint serum </td><td> Life Technologies </td><td> 12483020 </td><td></td></tr><tr><td> 0,25% trypsin-EDTA </td><td> Life Technologies </td><td> 25.200-72 </td><td></td></tr><tr><td> Hanks balancerede saltopløsning </td><td> Sigma Aldrich </td><td> H6648 </td><td></td></tr><tr><td> Lipofectamin 2000 </td><td> Life Technologies </td><td> 11668-019 </td><td></td></tr><tr><td> Nikon Eclipse TE2000-5 </td><td> Nikon Instruments Inc. </td><td></td><td></td></tr><tr><td> Xenolight D-luciferin </td><td> PerkinElmer </td><td> 122.796 </td><td> 40 mg / ml i PBS </td></tr></tbody></table>

molecular imaging to target transplanted muscle progenitor cells

Duchenne muskeldystrofi (DMD) er en alvorlig genetisk neuromuskulær lidelse, der påvirker 1 på 3.500 drenge, og er kendetegnet ved progressiv muskel degeneration 1, 2. Hos patienter, bliver evnen til residente muskel satellit celler (SCS) at regenerere beskadigede myofibre stadig ineffektiv 4. Derfor transplantation af muskel progenitorceller (MPL) / myoblaster fra raske personer er en lovende terapeutisk tilgang til DMD. En væsentlig begrænsning i anvendelsen af ​​stamcellebehandling er imidlertid en mangel på pålidelige billeddannende teknologier til langtidsovervågning af implanterede celler, og for at evaluere dens effektivitet. Her beskriver vi et ikke-indtrængende, real-time fremgangsmåde til at evaluere effekten af ​​myoblast transplantation. Denne fremgangsmåde drager fordel af en samlet fusion reportergen bestående af gener (ildflue-luciferase [udsving], monomert rødt fluorescerende protein [mrfp] og sr39 thymidinkinase [sr39tk]), hvis expression kan afbildes med forskellige afbildningsmodaliteter 9, 10. En række forskellige billeddannende modaliteter, herunder positronemissionstomografi (PET), single-photon emission computed tomography (SPECT), magnetisk resonansbilleddannelse (MRI), optisk billeddannelse og højfrekvens 3D-ultralyd er nu tilgængelige, hver med unikke fordele og begrænsninger 11. Bioluminescens imaging (BLI) undersøgelser, for eksempel har den fordel, at relativt lave omkostninger og højt gennemløb. Det er af denne grund, at det i denne undersøgelse, har vi gør brug af ildflue-luciferase (udsving) reportergen sekvens indeholdt i fusionsgenet og bioluminescens billeddannelse (BLI) for den korte lokalisering af levedygtige C2C12 myoblaster efter implantation i en mus model for DMD (muskeldystrofi på X-kromosomet [MDX] mus) 12-14. Vigtigere, BLI giver os et middel til at undersøge kinetikken af ​​mærkede Middelhavspartnerlandene post-implantation, og vil være nyttigt at spore celler gentagne gange over time og efterfølgende migration. Vores reporter gen tilgang yderligere giver os mulighed for at flette flere billeddiagnostiske metoder i en enkelt levende emne; givet tomografisk natur, fine rumlige opløsning og evne til at skalere op til større dyr og mennesker 10,11, vil PET danner grundlag for det fremtidige arbejde, som vi antyder kan lette hurtig translation af metoder udviklet i celler til prækliniske modeller og kliniske anvendelser.

Ved 50-60% konfluens blev C2C12 myoblaster transient transficeret med det ovennævnte fusionsprotein reportergenkonstruktionen sammensat af ildflue-luciferase [udsving], monomert rødt fluorescerende protein [mrfp] og sr39 thymidinkinase [sr39tk] (figur 1A). Transfektionseffektivitet blev beregnet via fluorescensmikroskopi (fig. 1B, C), der gør brug af mrfp sekvensen i vores reporterkonstruktion. Cell overlevelsesevne blev ikke påvirket af mærkning...

Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

En ikke-invasiv måde at evaluere succes myoblast transplantation er beskrevet. Fremgangsmåden drager fordel af en samlet fusion reportergen bestående af gener, hvis ekspression kan afbildes med forskellige billeddannende modaliteter. Her vil vi gøre brug af en<em&gt; Udsving</em&gt; Reportergen sekvens til målceller via bioluminescens billeddannelse.

Molekylær Imaging til TARGET Transplanterede Muskel progenitorceller

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

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