Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells 英文参考文献.docVIP

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Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells 英文参考文献.doc

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Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells 英文参考文献

ExpressionofPrionProteininMouseErythroid ProgenitorsandDifferentiatingMurineErythroleukemia Cells MartinPanigaj,HanaGlier,MarcelaWildova,KarelHolada* InstituteofImmunologyandMicrobiology,FirstFacultyofMedicine,CharlesUniversityinPrague,Prague,CzechRepublic Abstract Prion diseases have beenobserved toderegulate thetranscription of erythroid genes, andprion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC)inerythropoiesis,westudiedtheprotein’sexpressiononmouseerythroidprecursorsinvivoandutilizedaninvitro modeloftheerythroiddifferentiationofmurineerythroleukemiacells(MEL)toevaluatetheeffectofsilencingPrPCthrough RNA interference. The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flowcytometry,westernblotanalysisandquantitativeRT-PCR.ThesilencingofPrPCexpressioninMELcellswasachievedby expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrPC expression in differentiatingerythroidprecursorswasdetectedbothinvivoandinvitro,suggestingPrPC’simportancetotheearlystages ofdifferentiation.Theupregulationwashighestonearlyerythroblasts(1620063700PrPC/cell)andwasfollowedbythe gradualdecreaseofPrPClevelwiththeprecursor’smaturationreaching4706230PrPC/cellonmostmatureCD712Ter119+ smallprecursors.Interestingly,thedownregulationofPrPCproteinwithmaturationofMELcellswasnotaccompaniedby thedecreaseofPrPmRNA.Thestableexpressionofanti-PrnpshRNAmirinMELcellsledtotheefficient(.80%)silencingof PrPClevels.Cellgrowth,viability,hemoglobinproductionandthetranscriptionofselecteddifferentiationmarkerswerenot affectedbythedownregulationofPrPC. Inconclusion,theregulationofPrPCexpressionindifferentiatingMELcellsmimics thepatterndetectedonmouseerythroidprecursorsinvivo.DecreaseofPrPCproteinexpressionduringMELcellmaturation isnotregulatedontranscriptionallevel.TheefficientsilencingofPrPClevels,despitenotaffectingM