Name: intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice
Uploaded: 2018-02-10T13:00:00
Description: Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Dit werk werd gesteund door de core subsidie ontvangen van de afdeling van de biotechnologie, van de Indiase regering aan de nationale Instituut van immunologie, New Delhi. Dr. Bhattacharjee de huidige adres is een divisie van gastro-enterologie, leverziekten en voeding, Children's Hospital Los Angeles.

Procedures gepresenteerd in dit protocol zijn goedgekeurd door de institutionele dier ethisch comité van het nationale Instituut van immunologie, New Delhi. De seriële referentienummer van de goedkeuring is IAEC #319/13.
Opmerking: Er zijn uitstekende middelen op algemene chirurgie procedures20 en specifieke protocollen voor knaagdieren chirurgie21. Voor degenen die dierlijke chirurgie doen voor de eerste keer, is het aanbevolen om uit...

<ol>
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Gedeeltelijke hepatectomy is een gevestigde techniek voor behandelende lever regeneratie en buitensporige hepatectomy is gemeld na te bootsen de AHF model. Dierlijke modellen van AFH behoren knaagdieren, met name muizen, tot de meest onderzochte model. Om op te halen van een model van de lever schade bij muizen, tot een 70% hepatectomy is gemeld met een goede overleving tarief25,26. Echter in naakt en andere immunodeficiëntie muis, een 70% hepatectomy is gemeld ...

Op dit moment wordt hepatocyte transplantatie voorgesteld als een alternatief voor hele orgaantransplantatie voor behandelen patiënten met ernstige leveraandoeningen. Het is van mening dat patiënten op hele orgaantransplantatie1kan overbruggen. Naast de allogene hepatocyten2, worden xenogene hepatocyten3 en hepatocyten afgeleid van stamcellen4 ook onderzocht in diermodellen. In deze context, de homing en engraftment is potentieel van de getransplanteerde cellen in de ontvanger een belangrijk criterium voor celtherapie gebaseerd in acute hepatische insufficiëntie (AHF).
Voor het onderzoeken van de transplantatie van hepatocyten of hepatocyte-achtige cellen5, is AHF gemaakt in een diermodel door chirurgische6 of farmacologische7 procedures, gevolgd door het transplanteren van de cellen. Te maken van een dierlijk model van AHF door farmacologische reagentia, vele hepatotoxine zoals d-galactosamine8paracetamol9tetrachloorkoolstof10, thioacetamide11, Concanavalin A12, lipopolysaccharide13 , enz., zijn gebruikt. In deze lijst elk reagens genereert een unieke reeks van functies voor AHF, maar helaas geen enkele reagens bootst de menselijke AFH. Bovendien, de AFH geïnduceerd door hepatotoxine lang duurt, waardoor dieren onder chronische stress, en reproduceerbare resultaten zijn moeilijk te verkrijgen.
Aan de andere kant, de chirurgische ingreep van gedeeltelijke hepatectomy (PHx) is vaardigheid afhankelijk, en reproduceerbare resultaten zijn eenvoudig te verkrijgen na het ontwikkelen van de vereiste vaardigheden. Om te induceren AHF door chirurgische ingreep alleen, is resectie van meer dan 70% van de lever vereist; echter getransplanteerde minder dan een 70% hepatectomy kan nog steeds worden gebruikt om de studie van engraftment en verspreiding van cellen in de lever voor het analyseren van hun therapeutische capaciteit tijdens leverschade14. De transplantatie van hepatocyten geweest uitgevoerd post hepatectomy via het buikvlies15, staart ader16, hepatische ader17of de milt18. Op dit moment, zijn hepatische ader infusie en intrasplenic transplantatie van hepatocyten de aangewezen procedures, zoals ze gemakkelijker zijn te reproduceren.
In dit document, hebben we een procedure voor een gedeeltelijke hepatectomy van 30% in KNIPOOG beschreven. SCID (NOD. CB17-Prkdcscid/J) muizen waarin de linker kwab van de lever is weggesneden. Het wordt gevolgd door transplantatie van 0,2 miljoen GFP waarin muis (C57BL/6-Tg (UBC-GFP) 30Scha/J) hepatocyten evenals menselijke oorsprong NeoHep19 in de milt. Deze procedure leidt tot engraftment van de getransplanteerde cellen in de lever. Deze procedure is de minst invasieve en een minimaal pijnlijke techniek.

<table border="0" cellpadding="0" cellspacing="0" width="1002">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Gas Anesthesia System</td>
			<td style="width:255px;">Ugo Basile; Italy</td>
			<td style="width:119px;">211000</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weighing machine</td>
			<td style="width:255px;">Goldtech ; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biological safety cabinet ( Class I)</td>
			<td style="width:255px;">Kartos international;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hair Trimmer</td>
			<td>Panasonic ;&#160; Japan&#160;</td>
			<td style="width:119px;">ER-GY10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight operating scissor with sharp /sharp blades</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps with Serrations</td>
			<td style="width:255px;">Major Surgicals; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders&#160; straight &#38; curved&#160;</td>
			<td>Mercian ;&#160; England&#160;</td>
			<td style="width:119px;">BS-13-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml insulin syringe with 30G *5/16 needles&#160;</td>
			<td>Dispo Van; India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringe with 26 G * 1/2 needle</td>
			<td>BD ; US&#160;</td>
			<td>REF 303060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon Threads&#160;&#160;</td>
			<td style="width:255px;">Mighty ; India</td>
			<td style="width:119px;"></td>
			<td>(1-0) Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MERSUTURES 4-0 Sterilised Surgical Needled Suture</td>
			<td style="width:255px;">Ethicon, Johnson &#38; Johnson, India</td>
			<td style="width:119px;">NW 5047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRUGUT 76 cm 4-0 absorbable surgical suture</td>
			<td>Sutures India Pvt. Ltd; India</td>
			<td>SN 5048</td>
			<td>Sterilised Surgical Needled Suture Catgut Chromic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Cotton Buds</td>
			<td style="width:255px;">Pure Swabs Pvt Ltd ;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Local Procurement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Surgical Tape</td>
			<td style="width:255px;">3M India ; India</td>
			<td style="width:119px;">1530-1</td>
			<td>Micropore Surgical Tape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Microtome</td>
			<td style="width:255px;">Histo-Line Laboratories, Italy</td>
			<td style="width:119px;">MRS3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shandon Cryotome E Cryostat</td>
			<td style="width:255px;">Thermo Electron Corporation ; US</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Confocal laser scanning microscope</td>
			<td style="width:255px;">Carl Zeiss ; Germany&#160;</td>
			<td style="width:119px;">LSM 510 META</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Bright Field Microscope</td>
			<td style="width:255px;">Olympus, Japan</td>
			<td style="width:119px;">LX51</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Automated analyser</td>
			<td>Tulip, Alto Santracruz, India</td>
			<td style="width:119px;">Screen Maaster 3000</td>
			<td>Biochemical analyser for liver functional test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Flow Cytometer</td>
			<td style="width:255px;">BD ; US&#160;</td>
			<td style="width:119px;">BD FACSverse</td>
			<td>Assesment of presence of cells post transplantation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veet hair removal cream&#160;</td>
			<td>Reckitt Benckiser , India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FORANE</td>
			<td style="width:255px;">Abbott ; US</td>
			<td style="width:119px;"></td>
			<td>isoflurane USP 99.9%&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taxim</td>
			<td style="width:255px;">AlKem ; India</td>
			<td style="width:119px;"></td>
			<td>cefotaxime sodium injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution&#160;</td>
			<td style="width:255px;">Win-Medicare;&#160; India</td>
			<td style="width:119px;"></td>
			<td>Betadine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Paraformaldehyde</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">GRM 3660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">12200-036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Sucrose</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Tissue-Tek</td>
			<td style="width:255px;">Sakura; US</td>
			<td>25608-930</td>
			<td>O.C.T compound</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAPI</td>
			<td style="width:255px;">Himedia; India</td>
			<td style="width:119px;">MB 097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Albumin goat Polyclonal</td>
			<td style="width:255px;">Thermo Scientific,Pierce, US</td>
			<td style="width:119px;">PA126081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-connexin 32/GJB1 Polyclonal</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab64609-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">antiGFP rabbit polyclonal&#160;</td>
			<td style="width:255px;">Santa Cruz biotechnology; US</td>
			<td style="width:119px;">SC 8334</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 donkey anti-goat</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11058</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 488 donkey anti-sheep</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A11015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Alexa Fluor 594 chicken anti rabbit</td>
			<td style="width:255px;">&#160;Molecular Probes , Thermo Fisher Scientific ;&#160; US</td>
			<td style="width:119px;">A21442</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:411px;">Goat anti rabbit IgG HRP</td>
			<td style="width:255px;">Invitrogen, Thermo Fisher Scientific; US&#160;</td>
			<td>65-6120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">anti-Ki67 antibody</td>
			<td style="width:255px;">abcam, UK</td>
			<td style="width:119px;">ab15580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Antigen Unmasking Solution, Citric acid base</td>
			<td style="width:255px;">Vector laboratories, US</td>
			<td style="width:119px;">H-3300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ProLong Diamond antifade mountant</td>
			<td style="width:255px;">Life technologies, Thermo Fisher scientific ; US</td>
			<td style="width:119px;">P36966</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGOT (ASAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">SGPT (ALAT) KIT</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Alkaline Phosphatase Kit (DEA)</td>
			<td style="width:255px;">Coral Clinical System, India</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hematoxylin Solution, Mayer&#39;s</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">MHS16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Eosin Y solution, alcoholic</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">HT110132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DPX Mountant&#160;</td>
			<td style="width:255px;">Sigma ; US</td>
			<td align="right" style="width:119px;">6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Melonex (Pain Killer)</td>
			<td style="width:255px;">Intas Pharmaceuticals Ltd; India</td>
			<td style="width:119px;"></td>
			<td>Meloxicam injection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">DAB enhanced liquid substrate system tetrahydrochloride</td>
			<td style="width:255px;">Sigma ; US</td>
			<td style="width:119px;">D3939</td>
			<td></td>
		</tr>
	</tbody>
</table>

intrasplenic transplantation of hepatocytes after partial hepatectomy in nod.scid mice

Gedeeltelijke hepatectomy is een veelzijdig en reproduceerbare methode lever regeneratie en het effect van cellen gebaseerde therapeutics in verscheidene pathologische condities te studeren. Gedeeltelijke hepatectomy vergemakkelijkt ook de verhoogde engraftment en de verspreiding van getransplanteerde cellen door versnelde neovascularization en cel migratie naar de lever. Hier beschrijven we een eenvoudig protocol voor het uitvoeren van 30% hepatectomy en transplantatie van cellen in de milt van een nietzwaarlijvige diabetische/ernstige gecombineerde immunodeficiëntie knik. SCID (NOD. CB17-Prkdcscid/j) muis.
In deze procedure zijn twee kleine incisies gemaakt. De eerste incisie wordt bloot en resect de linker kwab van de lever, en een andere kleine incisie wordt gemaakt om de milt voor de intrasplenic transplantatie van cellen bloot te stellen. Deze procedure is niet vereist geen gespecialiseerde chirurgische vaardigheden en deze kan worden voltooid in 5-7 minuten met minder stress en pijn, sneller herstel en beter overleven. We hebben aangetoond dat de transplantatie van hepatocyten geïsoleerd van een groen fluorescente proteïne (GFP) uiting van de muis (transgene C57BL/6-Tg (UBC-GFP) 30Scha/J), evenals de hepatocyte als cellen van menselijke oorsprong (NeoHep) in gedeeltelijk hepatectomized knik. SCID muizen.

Hepatocyte proliferatie na 30% gedeeltelijke hepatectomy: De proliferatie van hepatocyten in de resterende lever na 30% hepatectomy werd onderzocht door immunohistochemische (IHC) kleuring voor een cel proliferatie marker, Ki-67. Eendaagse post hepatectomy, de muizen waren euthanized, de resterende lever kwabben waren weggesneden en paraffine secties werden verkregen. De secties zijn gekleurd met Ki-67 antilichaam, gevolgd door etikettering met horseradish peroxidase (HRP) geconjugeerd secundair antilich...

Watch this Scientific Journal Video about Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice at JoVE.com

Intrasplenic Transplantation of Hepatocytes After Partial Hepatectomy in NOD.SCID Mice

We hebben een protocol voor het uitvoeren van gedeeltelijke hepatectomy (PHx) en cel transplantatie via milt in KNIPOOG beschreven. SCID (NOD. CB17-Prkdcscid/j) muizen. In dit protocol wordt een incisie gemaakt bloot en resect de linker kwab van de lever, gevolgd door een andere snede voor intrasplenic transplantatie van cellen.

Intrasplenic transplantatie van hepatocyten na gedeeltelijke Hepatectomy in KNIPOOG. SCID muizen

Partial hepatectomy is a versatile and reproducible method to study liver regeneration and the effect of cell based therapeutics in various pathological ...

Research

JoVE Journal

Medicine

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Chirurgische ingrepen voor een Rat Model van deelspoelen orthotope levertransplantatie met hepatische arteriële reconstructie

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Heterotopic Cervical Heart Transplantation in Mice

Heterotopische Cervical harttransplantatie in Muizen

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Visualization of Vascular and Parenchymal Regeneration after 70% Partial Hepatectomy in Normal Mice

Visualisatie van vasculaire en Parenchymale Regeneratie na 70% Gedeeltelijke hepatectomy in normale muizen

A modified silicone injection procedure was used for visualization of the hepatic vascular tree. This procedure consisted of in-vivo injection of the ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Opnieuw Arterialized Rat gedeeltelijke lever transplantatie met een in vivo vaartuig georiënteerde 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Transplantatie van stamcellen adipeus weefsel afkomstige blad om lekkage na gedeeltelijke Colectomy in een Rat-Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Lentivirale Vector-gemedieerde gentherapie van hepatocyten Ex Vivo voor autologe transplantatie in varkens

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Generatie van een rat model van acuut leverfalen door het combineren van 70% partiële Hepatectomie en Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

Verminderde complicaties na arteriële heraansluiting in een ratmodel van orthotopische levertransplantatie

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Intramyocardtransplantatie van MSC-Loading Injecteerbare Hydrogels na myocardinfarct in een Murine-model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...