细胞生物学作业.docx

细胞生物学作业.docx

《细胞生物学作业.docx》由会员分享，可在线阅读，更多相关《细胞生物学作业.docx（27页珍藏版）》请在冰豆网上搜索。

细胞生物学作业

Foeturnedfriend:

multiplefunctionalrolesattributabletohyper-activating

stemcellfactorreceptormutantinregenerationofthehaematopoieticcell

compartment

S.Pati*,1,O.P.Kalra†andA.Mukhopadhyay*

*StemCellBiologyLaboratory,NationalInstituteofImmunology,ArunaAsafAliMarg,NewDelhi,India,and†DepartmentofMedicine,University

CollegeofMedicalSciencesandGuruTegBahadurHospital,UniversityofDelhi,NewDelhi,India

Received1June2010;revisionaccepted2July2010

Abstract

Objectives:

Stemcellfactorreceptor,c-kit,is

consideredtobethemastersignallingmoleculeof

haematopoieticstemcells.Itdevelopstheorchestral

patternofhaematopoieticcelllineages,seenbyits

varyingdegreeofomnipresenceinprogenitors,lineage

committedandmaturecells.Wehaveinvestigated

theeffectofover-expressingc-kitonearly

recoveryofthehaematopoieticcompartment,inirradiated

hosts.

Materialsandmethods:

Normalbonemarrowcells

（BMCs）weretransfectedwithKitwt（wild-typec-kit）

oritsvariantKitmu（asp814tyr）byelectroporation.

Lethallyirradiatedmiceweretransplantedwithnormal

ortransfectedcongeneicBMCs.Theeffectof

ectopicexpressionofc-kitonhaematopoieticcell

recoverywasdeterminedbyanalysingdonor-derived

cells.Furthermore,effectsofbothtypesofc-kitoverexpression

onprogenitorandlineage-committedcells

wereexaminedbyflowcytometricanalysisofSca-1

andlineage-committed（Lin+）cellsrespectively.

Results:

Hyper-activatingKitmusignificantlyimproved

recoveryofthehaematopoieticsystemin

irradiatedhosts.Invivoresultsshowedthatthe

donor-derivedc-kit+cellpopulationwasincreasedto

morethan3-foldinthecaseofKitmu-transfectedcells

comparedtonormalandKitwtover-expressing

BMCs.Ingeneral,survivalofprogenitorand

committedcellwasimprovedintheKitmu

over-expressingsystemcomparedtotheothertwo

cohorts.

Conclusion:

Theseresultssuggestthatrecruitment

ofthehyper-activatingvariantofc-kit（Kitmu）leadto

earlyrecoveryofthebonemarrowoflethally

irradiatedmice.

Introduction

Haematopoieticstemcells（HSCs）areembodiedwith

immensecapabilitytoconstructacompletehaematopoietic

compartmentfromasinglecell,whichactsastheprimitive

powerhouse（1,2）.Radiation-induceddamageofthe

bonemarrow（BM）leadstomyelodysplasia,resulting

fromdeathofprogenitors,withconsequentlossoffunctional

cells.Thecurrentapproachtosuccessfulmanagement

oftreatingmalignancyincludesradiationand⁄or

chemotherapy.Thismayresultindepletionofstemcells,

organfailureandlateeffectsthatmightcontributeto

malignanttransformation（3）.

Patientsundergoingradiationaregivenhaematopoietic

celltherapyfromthreemajorsources:

（i）autologous⁄allogeneic

BM,（ii）cytokinemobilizedautologousperipheral

blood,andrecently（iii）cordblood.Thesesourcesofcells

haveseverallimitationssuchasimproperHLA-matched

donorcells,requirementofimmunosuppressanttreatment,

recurrenceoftumoursandrequirementofhighdosageof

cellsfortransplantation.Theabovelimitationsareclinically

manifestedasearlyeffects（forexample,mucositis,

graft-versus-hostdisease,haemorrhagiccystitis,lung

injuryandveno-occlusivedisease）andlateeffects（for

example,musculoskeletaleffects,oculareffects,endocrine

effectsandneurocognitiveandneuropsychological

effects）,thusdemandingfasterrecoveryofthehaematopoietic

system.

Correspondence:

A.Mukhopadhyay,StemCellBiologyLaboratory,

NationalInstituteofImmunology,ArunaAsafAliMarg,NewDelhi-

110067,India.Tel.:

+91-11-26703781;Fax:

+91-11-26702125;E-mail:

ashok@nii.res.in

1Presentaddress:

DepartmentofNeuroscience,SchoolofMedical

Sciences,UniversitySainsMalaysia,Malaysia.

10_2010BlackwellPublishingLtd.

CellProlif.,2011,44,10–18doi:

10.1111/j.1365-2184.2010.00713.x

Earlyrecoveryofthehaematopoieticsystemfrom

lethalirradiationisstrictlylimitedduetoG0-silenceor

quiescencenatureofHSCs.Hence,analternativestrategy

isexpectedtobebeneficialbyengineeringdormanthaematopoietic

cellsforinitiationofearlysignallingevents.

AlthoughmanyHSC-specifictranscriptionfactors,growth

factorreceptorsandcorrespondingligandsareknownto

activatehaematopoieticcells,recruitmentofamastersignalling

switchwouldensurestage-specificregenerationof

cells（4–6）.

Previousstudieshaveshownthatviralvector-based

genedeliveryinHSCshasmanydrawerbacks（7–9）.

PrimitiveHSCs（Lin）Sca-1+c-kit+;LSK）,whichare

only0.5%oftotalBMcells,havedenselypacked

DNAinsideintactnuclearmembranes.Thisrenders

themdifficulttotransfectbyaviral-mediatedgene

transfermethod.Thesecondobstructionisseenas

post-maintenanceofthesemodifiedpopulationsinvivo

forshort-termaswellaslong-termgeneexpression,as

aresultofcompetitionbetweentransducedandnontransduced

cells.Thishasbeenshowninthecaseof

patientswithchronicgranulomatousconditionsinfused

withgp91phox-transducedpurifiedCD34+peripheral

bloodcells（7）.Thethirdandmostsevereproblem

associatedwithviralgenetherapyisdevelopmentof

oncogenesisarisingfrominsertionalmutagenesis.Many

genetherapystudieshaveshowndevelopmentof

leukaemiainpatientswhohadundergonetreatmentfor

severecombinedimmunodeficiency（8,9）.

Consideringtheaboveshortfallsofvirus-mediated

genetransfer,weproposeaprotocolofelectroporationmediated,

transientover-expression,ofamastersignal

transducerc-kitwtanditshyper-activatingvariant[Kitmu

（asp814tyr）]toreconstitutethehaematopoieticsystem.

Theelectroporationmethodisknowntoinduceless

celldeathascellsdonotexperiencechemicaltoxicity

asinthecaseofchemical-mediatedtransfection（10–

12）.Earlierstudieshaveconfirmedc-kitwtasthekey

receptorforgrowthandsurvivalofHSCs（13,14）.

Undernormalcircumstances,c-kitisactivatedbyits

ligandstemcellfactor（SCF）.Inhumans,constitutive

activationofc-kitinneoplasticmastocytosishasbeen

showntobypassSCFinterferenceindownstreamsignalling

duetoclusteringofgain-of-functionmutation

Asp816toValintheKitactivationdomain（15）.

Recently,wehavepredictedthatcertainactivationloop

residuesinc-kitarecrucialforinteractionofkinases

withShp-1fordestabilization.Pointmutationin

stretchesofLys818toSer821andThr847toGlu849

intheKitcatalyticdomainmayleadtogenerationofa

hyperactivevariantofc-kit（16）.Inthemouse,Asp814

toTyrmutationoftheKitactivationloopresultsin

over-expressionofkinasesasbydegradationoftyrosine

phosphatase（Shp-1）（17）.HyperactiveKitkinase

hasbeenreportedtobeassociatedwithconstitutive

activationofphosphoinositide-3kinase（PI3kinase）,

signaltransducerandactivatoroftranscription3

（STAT3）,andnuclearfactorkappaB（NF-jB）signalling

pathways[16–18].Cellsaffectedwerealsoindependent

ofSCFforgrowth（18）.Theseresultssuggest

thatrecruitmentofahyper-activatingmutantlikeKitmu

（asp814tyr）wouldinfluencedownstreamsignallingin

lethallyirradiatedhoststofacilitatehaematopoiesis.

Materialsandmethods

Animals

Six-toeight-week-oldC57BL⁄6J[Ptprcb（Ly5.2）]and

BL6.SJL[Ptprcc（Ly5.1）]micewereusedinthisstudy.

MicewereobtainedfromtheJacksonLaboratory（Bar

Harbor,Maine,USA,http:

//www.jax.org）andmaintained

inourinstitute’sexperimentalanimalfacility.Duringthe

experiments,micewerekeptinanisolatorandfedautoclaved

acidifiedwaterandirradiatedfoodadlibitum.All

experimentsusingmicewereconductedaccordingtothe

proceduresapprovedbytheInstitutionalAnimalEthics

Committee.

IsolationofBMcells

BMCswereisolatedbyflushingtwotibiaandtwofemurs

from6-to8-week-oldLy5.1mice,anderythrocyteswere

lysedbytreatmentwithGey’ssolution（19）.Cellsuspension

wasincubatedinatissuecultureplatefor12hto

removeadheredstromalcells.Priortoelectroporation,

non-adherentviablecellswerecountedusingthetrypan

bluedyeexclusionmethod.

ElectroporationofBMcells

BMCswereculturedfor12hat37_CinaCO2incubator.

Electroporationofcells（10・106percuvette）wasperformed

usingaGenePulserIIBioradelectroporator（Hercules,

CA,USA）at1.7kV⁄cmcurrentand400lspulse

length.Acustom-mademinipulsechamber（withcapacity

ofhighcelldensity）havingrectangularstainlesssteel

electrodes（4mm）wasusedforelectroporation.Polyethylene

glycol-purifiedpcDNA3.1-Kitconstructswere

dilutedinelectroporationbuffer[10mMHEPES,IMDM

supplementedwith5%FCSand5%FBS]ataconcentration

of50–100lgDNA⁄ml.Toavoidpost-pulseapoptosis,

electroporatedcellswerewashedandincubatedat

37_Cinthe5%CO2incubatorfor15min.Incubationof

post-pulsedcellsisbelievedtofacilitateresealingofpores

（10,11）.

_2010BlackwellPublishingLtd,CellProliferation,44,10–18.

Haematopoieticrecoverybyhyper-activatingc-kit11

CultureofLy5.1BMCs

ElectroporatedcellswereculturedinIscove’smodified

Dulbecco’smedium（IMDM）,supplementedwith10%

FCS,50ng⁄mlmurineSCFand10ng⁄mlinterleukin-3

（IL-3）（PeproTechAsia,Rehovot,Israel,http:

//www.

）.Cellswereculturedfordifferenttimesat

37_Cina5%CO2incubatorandviablecellnumberwas

determined.

Flowcytometry

Cellswerelabelledwithantibodiesandanalysedaccording

tomethodsdescribedinourpreviousstudy（20）.

Antibodiesusedherewereanti-:

Sca-1⁄FITC,c-kit⁄

phycoerythrin-Cy5（BDPharmingen,SanJose,CA,

USA）,CD45.1⁄PE-Cy5andCD16⁄32（eBiosciences,San

Diego,CA,USA）.Lineagea