Name: tumor engraftment in a xenograft mouse model of human mantle cell lymphoma
Uploaded: 2018-03-30T12:30:00
Description: Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

Dette arbejde blev støttet af Ligue Genevoise contre le Cancer, Fondation Dr. Dubois Ferriere Dinu-Lipatti, Oncosuisse KPS-OCS, OCS-02260-08-2008-2914-02-2012, og Swiss National Science Foundation Grant 31003A_156760 og 310030-153456.

De menneskelige blodprøver blev brugt efter procedurer, der er godkendt af de lokale etiske og menneskelige eksperimenter udvalg af Genève University Hospital.
Animalske procedurer blev udført i overensstemmelse med den institutionelle etiske udvalg af Animal Care i Genève, Schweiz og den kantonale Veterinærkontoret (nummer for tilladelsen: 26-GE-15).
1. forberedelse af primære perifert blod mononukleære celler (PBMC) af tæthed Gradient adskillelse
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Kliniske forsøg er muligt for lægemidler, der er på et avanceret udviklingstrin, men kan ikke anvendes for drug discovery. Bestræbelser på at udvikle animalske modtagere for humane celle xenotransplantation for at teste tidlige fase medicin har længe været forfulgt. Her præsenterer vi en dyremodel, der undgår menneskelige graft afvisning og kan etablere en model for menneskelige sygdomme, såsom MCL. Dette er i øjeblikket en topmoderne xenograft model til at studere mekanismerne af menneskelige tumor engraftmen...

Lymfocytter af natur spiller en stor rolle i immunforsvaret overvågning, og lymfocyt handel er et kritisk skridt i montering antigen specifikke immunitet1,2. Denne proces omfatter overførsel af naive T-lymfocytter fra thymus at blodet, og derfra til sekundære lymfoide organer, herunder lymfeknuder, Peyerske plaques eller milten, hvor de mødes beslægtet antigener. B-lymfocytter differentiere i knoglemarven og overflytte som naiv celler i follikler af sekundære lymfoide organer3. Nogle af disse B-celler binder antigen med deres receptor og aktiveres ved specifikke T-celler. Spredning og differentiering af disse B-celler skubber de ikke-aktiveret, naive B-celler i zonen kappe af hårsækken. Aktiverede celler kan derefter differentiere i hukommelse B-celler, som patruljerer kroppen, eller modnes til immunoglobulin secernerende plasmaceller, der vandrer til knoglemarven4.
MCL opstår når naive B-lymfocytter i zonen kappe forvandle sig til en tumor. Disse lymfom celler er placeret i mikromiljø lymfoide organer og formere sig uafhængigt af specifikke T-lymfocytter kontrol. Men på et bestemt tidspunkt af tæthed de flygte fra dette niche og recirkulere i blodbanen i søgen efter nicher i andre organer. I betragtning af kompleksiteten af adhæsionsmolekyler og promiskuitet kemokiner og deres receptorer, mekanismen af dette cellulære menneskehandel i vivo er dårligt forstået og derfor hæmmer terapi. Nye metoder er nødvendige for effektivt blokere denne migration processen for at forhindre lymfom B celler fra at nå nye microenvironments.
MCL er en af de mest vanskelige at behandle B celle maligniteter. Udviklingen af en neoplastiske Fænotypen af MCL er resultatet af en omstændelig kaskade, karakteriseret ved erhvervelse af unikke biologiske egenskaber. På tidspunktet for diagnosen, de fleste patienter (70%) allerede er til stede med en dissemineret sygdom, med et flertal af tilfælde udstiller extranodal inddragelse i milt, knoglemarv og/eller mavetarmkanalen5,6. I behandlede patienter er tilbagefald af resistente tumorer inden for et par år fælles, selvom konventionel kemoterapi inducerer høje remission priser på kort sigt7,8. Her præsenterer vi en ny sygdom model, der kan hjælpe med at forstå MCL formidling og dets underliggende Biologi: vi etableret en menneskelig MCL xenograft musemodel, der stammer fra primær tumorceller af patienter. Vi håber, at denne model vil hjælpe med at udvikle terapeutiske strategier mod MCL formidling, og muligvis give nye kliniske perspektiver for optimal diagnose og behandling af recidiverende patienter.

<table>
	<tbody>
		<tr>
			<td>Ficoll-paque media</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td>for separation of mononuclear cells</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640</td>
			<td>Gibco-Life technologies</td>
			<td>61870-010</td>
			<td>for dilution of blood sample</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>washing of cells</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A8412</td>
			<td>for preparation of PBS with 1% BSA used in washing cells during isolation</td>
		</tr>
		<tr>
			<td>CD19-APC700</td>
			<td>Beckman Coulter</td>
			<td>B49212</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-APC</td>
			<td>Beckman Coulter</td>
			<td>A21693</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD20-ECD</td>
			<td>Beckman Coulter</td>
			<td>IM3607</td>
			<td>human pan-B cell marker</td>
		</tr>
		<tr>
			<td>CD5-PC 5.5</td>
			<td>Beckman Coulter</td>
			<td>PN A70203</td>
			<td>human T cell marker</td>
		</tr>
		<tr>
			<td>CD23-PE</td>
			<td>Pharmingen</td>
			<td>555711</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>CD45 KO</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td>Pan-leucocyte marker</td>
		</tr>
		<tr>
			<td>CD200-PE</td>
			<td>Pharmingen</td>
			<td>552475</td>
			<td>Cell surface protein typically absent in MCL</td>
		</tr>
		<tr>
			<td>NOD scid gamma (NSG) mice</td>
			<td>Charles River Laboratories</td>
			<td>5557</td>
			<td>used to develop MCL xenografts in this study</td>
		</tr>
		<tr>
			<td>Easy sep Human B cell enrichment kit</td>
			<td>Stem cell technologies</td>
			<td>19054</td>
			<td>used to enrich B cells to obtain pure cells for injecting into mice</td>
		</tr>
		<tr>
			<td>FACS</td>
			<td>Beckman Coulter</td>
			<td>Navios</td>
			<td>used to characterize MCL sample and to study the organs for MCL engraftment</td>
		</tr>
		<tr>
			<td>1X ammonium chloride potassium buffer</td>
			<td></td>
			<td></td>
			<td>red blood lysis buffer (NH4Cl 8,024 mg/l; KHCO3 1,001 mg/l; EDTA.Na2&#183;2H2O 3.722 mg/l )</td>
		</tr>
	</tbody>
</table>

tumor engraftment in a xenograft mouse model of human mantle cell lymphoma

B-lymfocytter er centrale aktører i immun celle omsætning og de primært hjem til og opholde sig i lymfoide organer. Mens normale B celler formere kun når stimuleret af T-lymfocytter, muterede B celler overleve og udvide selvstændigt i udefineret orgel nicher. Mantle celle lymfom (MCL) er en sådan lidelse, B-celle, hvor den mediane overlevelse af patienter er 4-5 år. Dette kræver brug af effektive mekanismer, som målsøgende og engraftment af disse celler er blokeret for at øge overlevelse og levetiden af patienter. Derfor, indsatsen for at udvikle en xenograft musemodel for at studere effekten af MCL therapeutics ved at blokere den homing mekanisme i vivo er af allerstørste betydning. Udvikling af animalske modtagere for humane celle xenotransplantation at teste tidlige fase narkotika har længe været forfulgt, som relevante prækliniske musemodeller er afgørende for skærmen nye terapeutiske agenter. Dette dyr model er udviklet til at undgå menneskelig graft afvisning og at etablere en model for menneskelige sygdomme, og det kan være et yderst nyttigt værktøj at studere sygdomsprogression af forskellige lymfom typer og udføre præklinisk afprøvning af kandidat narkotika for maligne hæmatologiske sygdomme som MCL. Vi etablerede en xenograft musemodel, der vil tjene som en fremragende ressource for at studere og udvikle nye terapeutiske tilgange til MCL.

Håndskriftet beskriver en optimeret protokol for en vellykket udvikling af en xenograft musemodel for engraftment MCL celler. Forberedelse af en ren celle population (i dette tilfælde MCL celler), er meget kritisk at udvikle vellykkede MCL xenografts. Figur 1 repræsenterer de forberedende skridt for mononukleære celler isolation fra MCL patientens blod af tæthed gradient adskillelse. Mononukleære celler behandles yderligere for at få rene B-celler ved ...

Watch this Scientific Journal Video about Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma at JoVE.com

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma (Video) | JoVE

Mantle celle lymfom (MCL) er en vanskelig at behandle B-celle sygdom og det er lige så vanskeligt at etablere en xenograft musemodel af primære MCL at studere og udvikle therapeutics. Her beskriver vi vellykket etablering af MCL xenografts i mus til at hjælpe med at forstå dens underliggende biologi.

Tumor Engraftment i en Xenograft musemodel af menneskelige Mantle celle lymfom

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