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1、中國(guó)農(nóng)業(yè)科學(xué)院碩士學(xué)位論文五指山豬近交系EG細(xì)胞建系影響因素研究姓名：劉嵐申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：動(dòng)物遺傳育種與繁殖指導(dǎo)教師：馮書堂20050601ABSTRACTAtotalof200fetuseswerecollectedfrom18wuZhiShaninbreedingPigs(wzsP)tostudyt1efhctorsinfluenceontheestablishmentofporcineembryonicgerm(EO)ce

2、llslinesThefactorsincludedfeedercells，conditionmedium(CM)，growthfactorsanddigestingenzymesMoreoverEGcellsevaluationwascarriedout(I)Porcineembryonicfibroblasts(PEF)wereobtainedfrom26～28dfetusesofWZSPtheycouldbepurifiedbyp

3、assagingPEFwerepassagedtoFlo，itWaSshownthatPEFcouldbeculturedlong—terminvitroandpreservedat——196“CThenormalkaryotypeofPEFwasfoundoutatF5bykaryotypeanalysisf2、T0study也eeffectoffeedercellsonporcineEGcellsculturedinvitro，53

4、fetuseswerecollectedfrom265dpregnantgilts33ofthemwereplatedonSTOfeedercellsand20werecoculturedwimPEFWhenusingthesamemediumporcinePGCderivedcoloniesculturedonSTOfeedercellscouldgrowwellThecoloniescoculturedwithPEFcoulddev

5、elopinF0，buttheydifferentiatedafterpassageSoPEFcouldnotmaintainthegrowthofporcineEGcells，whileSTOfeedercellsCOUIddo(3)Tostudytheeffectofconditionmedium(CM)onporcineEGcellsculturedinvitro，35fetuseswerecollectedfrom265dpre

6、gnantgilts15ofthemwereculturedinBRLCMand20wereincommonmedium(DMEM15％FCS2mmol／mlLglutamineOImmol／mlMEMnonessentialaminoacidsO1mmol／mlBmercaptoethanol1001U／mlPenicillin100pg／mlStreptomycin)TheresultsmanifestedthatbothBRLCM

7、andcommonmediumweresuitableforporcineEGcellsculturingandpassagingwhenhavingSTOfeedercellsSoBRL—CMcanbeusedtocultureporcineEGcells(4)TostudytheeffectofgrowthfactorsonporcineEGcellsculturedinvitro，78fetuseswerecollectedfro

8、m265dpregnantgilts35ofthemwereculturedinmediumsupplementedwithLIFSCFandbFGF(mediumII)and43wereculturedincommonmedium(mediumI)TheresultsshowedthatbothmediumIandmediumIIcouldbeusedculturingandpassagingporcineEGcellswhenhav

9、ingSTOfeedercells，butmedium11wasbetterthanmediumISoporcineEGcellscarlalsobeculturedinmediumwithoutgrowthfactors(5)TostudytheeffectofdigestingenzymesonpassageporcineEGcells，34fetuseswerecollectedfrom265dpregnantgiltsPGCsw

10、fftoisolatedandplmedonSTOfeedercellsOnpassaging，thecoloniesweredigestedbydispaseorO25％trypsinEDTATheresultwasthatrenascentcoloniesofEGcellscouldbeobtainedandpassagedatbothcasesItindicatedthatbothdispaseandO25％trypsin—EDT

11、AcanbeusedtopassageporcineEGcells(6)ThecoloniesofF3～F4EGceilswereidentifiedThemorphologyofporcinePGCderivedcolonieswasmultiplicitycellsweretightlypackedincoloniesandcoloniesedgewasplainThecolonieswereAKPpositive，reactedw

12、ithSSEA1antibodyandtheirimmunityfluorescencestainingofOct4waspositiveThecoloniesplacedinsuspensioncultureinmediumcontainingcalfserumwithoutgrowthfactorsdifferentiatedintosimpleembryoidbodies(EBs)Thesecellshavemanycharact

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