1、分類號密級y773533單位代碼：10019學(xué)號：B02198飛嚼摹r文等’博士學(xué)位論文云南矮馬與北京油雞成纖維細胞系建立與鑒定的研究ResearchontheEstablishmentandCharacterizingofYunnanPonyandBeijingYellowChickenfibroblastcelllines研究指導(dǎo)教師：—趙焦明熬援合作指導(dǎo)教師：申請學(xué)位門類級別：專業(yè)名稱：研究方向：所在學(xué)院：盔堂控基礎(chǔ)盞醫(yī)堂麴翅痘理

2、堂動物匡堂瞳二零零五年六月AbstractChinaasoneofthelargestanimalproducersintheworld，isrichinanimalgeneticresources，andtherearemanylocalbreedsscatteredthroughoutthecountryNevertheless，currentstatusforconservationofdomesticanimaldivemity

3、isnotoptimisticHigherleverofindustrializationisrealizedthroughraisingahigh—productivebreed(s)orhybrid(s)toreplacelocalbreedsDeclineanddeprivationofgeneticdiversityinpoultryandlivestockbreedstookplaceprogressivelyYunnanpo

4、nyandBeijingyellowchicken，whicharetwoimportantlocalbreedsinChinaweresampledandfibroblastcelllinesofthemwereestablishedandcharacterizedrespectivelyinthisresearch：1)TheearmarginaltissuesoftheYunnanponiesweresuccessfullycul

5、turedeitherbyprimaryexplanttechniqueorbycrudecollagenasetechniqueFibroblastcellswareseparatedbytrypsinizationandthreetimessubculturingMorphologyandanalysisofdynamicgrowthshowedthattherewerenosignificantmorphologicaldiffe

6、rencebetweencellsculturedwiththetwotechniquesafterthreepassages，andthepopulaliondoublingtime(PDT)ofcellsmadewiththetwotechniqueswere365hand315hrespectivelyTherefore，itWaSsuggestedthatwellcombinationofthetwotechniqueswasa

7、nefficientandfastmethodforthethintissueculturesuchasearmarginaltissueoftheYunnanponyandtheotheranimusMoreover’itWaSsignificantfortheconservationofnationalimportantdomesticanimalsandpoultrybreedsbeingindangerofextinctatth

8、ecellularlevelOtherwise，aBeijingyellowchickenembryofibroblastcelllineWaSsuccessfullyestablishedusingtheprimaryexplanttechniqueandpopulationdoublingtimeofthecensWaS397hAcertainextentnumberofceilsforthesetwobreedswereffeez

9、edandviabilityexaminationofthesecellsshowedthatviablerateswerehiigherthan90％beforefreezingandafterrecovery2)Observationsonmorphologyanalysisofkaryotype，isoenzymesoflactatedehydrogenaseandmalatedehydrogenaseforthetwonewly

10、establishedcelllineswereundertaken，andexpressionofrecombinantgreenfluorescenceproteininthecellswerecarriedoutTheresultsshowedthatforYunnanponythefrequencyofcellchromosomenumbertobe2n=64WaS929％：forBeijingyellowchicken，dip

11、loidcellsWeredominantof76％～88％Thebandingpatternsoftheisozymesofthetwoenzymeshadsignificantdifferenceamongthetwonewlyestablishedcelllinesandothercelllinesinourlaboratory；testsformicrobialcontaminationfrombacteriafungiormy

12、coplasmawerenegative；transferefficiencyfortheplasmidwashighof33％and25％respectivelyThetwonewlyestablishedcelllinesmaketheYunnanponyandBeUingyellowchickenbreeds，twonationalimportantgeneticresourcespreservedincelllevel，aswe

13、llaswillprovideanidealexperimentalmaterialforthegeneticstudiesonthesetwolocalbreeds31ThePrP。genesofYunnanponyandBeijingYellowchickenwereamplifiedbyPCR，andthesequenceswerecomparedwiththosepublishedofsomespeciesusingmolecu