Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes 英文参考文献.docVIP

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Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes 英文参考文献.doc

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Single-Cell Analysis of Ploidy and Centrosomes Underscores the Peculiarity of Normal Hepatocytes 英文参考文献

Single-CellAnalysisofPloidyandCentrosomes UnderscoresthePeculiarityofNormalHepatocytes FrancescaFaggioli1,2,PaoloVezzoni1,2*,CristinaMontagna3,4 1MilanUnit,IstitutodiRicercaGeneticaeBiomedica,ConsiglioNazionaledelleRicerche,Milan,Italy,2MedicalBiotechnologiesUnit,IstitutoClinicoHumanitas,Rozzano, Milan, Italy, 3Departments of Genetics, Albert Einstein College of Medicine, New York, New York, United States of America, 4Pathology, Albert Einstein College of Medicine,NewYork,NewYork,UnitedStatesofAmerica Abstract Polyploidizationisthemostwellrecognizedfeatureoftheliver.Yet,aquantitativeandbehavioralanalysisofcentrosomes andDNAcontentinnormalhepatocyteshasbeenlimitedbythetechnicalchallengesofmethodsavailable.Byusinganovel approachemployingFISHforchromosomes18,XandYweprovide,forthefirsttime,adetailedanalysisofDNAcopies duringphysiologicaldevelopmentintheliveratsinglecelllevel.WedemonstratethataneuploidyandunbalancedDNA contentinbinucleatedhepatocytesarecommonfeaturesinnormaladultliver.Despitethecommonbeliefthathepatocytes contain1,2ornomorethan4centrosomes,ourdoublestainingforcentrosomeassociatedproteinsrevealsextranumerary centrosomesinahighpercentageofcellsasearlyas15daysofage.Weshowthatinmurinelivertheperiodbetween15 daysand1.5monthsmarksthetransitionfromaprevalenceofmononucleatedcellstoupto75%ofbinucleatedcells.Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomesconvergewithseveralhepatocytesthatcontain3centrosomes;at1.5monthsthepercentageofcellswith3 centrosomesdecreasesconcomitantlywiththeincreaseofcellswithmorethan4centrosomes.Ouranalysisshowsthatthe extranumerarycentrosomesemergeinconcomitancewiththeprocessofbinucleationandpolyploidizationandmaintaina- tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal