ADPN基因構(gòu)建、蛋白表達(dá)及其對宮頸癌Hela細(xì)胞生長的影響.pdf

1、廈門大學(xué)碩士學(xué)位論文ADPN基因構(gòu)建、蛋白表達(dá)及其對宮頸癌Hela細(xì)胞生長的影響姓名：謝蓮英申請學(xué)位級別：碩士專業(yè)：細(xì)胞生物學(xué)指導(dǎo)教師：顏江華20070601GeneConstruction、ExpressionofADPNandItsInfluencesonHelaCellsGrowthAbstract：ThereductionofexpressionlevelofADPNhasbeenreportedtobeinverselyass

2、ociatedwithallincreasedriskofbreastcanc％endometrialcane％prostatecancercolorectalcancerandgastriccancerTheADPNlevelcallbeasanimportantindexfordetectionofdiseasesHowevertherelationshipbe鉚eenADPNandcervicalcancerhasnotbeenr

3、eportedTherefore，westudiedwhetherADPNinfluencestheproliferationofcervicalcancerHelacellsTheADPNgenewasobtainedbyoverlappingPCRtechniqueTheexpressionvectorpET22b()／ADPNWasconstructedandtransformedintoEcoliBL21(DE3)TheADPN

4、proteinwasexpressedasinclusionbodieswitlltheyieldofmorethan30％oftotalbacterialproteinsunderIPTGinductionTheexpressionproductswere。purifiedbyNi2NTAaffinitycolumn，SDS—PAGEanalysisshowedthattherelativemolecularweightofADPNi

5、s30kDawhichWasconsistentwiththetheoreticallypredictedvalueTheMTTassayshowedthatADPNcaninhibittheendothelialcellproliferationatsubphysiologicalconcentrationsTodetectthemechanismofADPNontumorcells，wedetectedtheexpressionof

6、ADPNreceptorsbyRTPCRandFITCfluorescencelabeledmethodinMGC803、BGC823、HT29、HelacellsMITassayanalyzedtheinfluenceofADPNontheproliferationrateofthetheseCanCercells，F(xiàn)lowcytometryanalyzedtheinfluenceofADPNonthecellcycle，RTPCRd

7、etectedtheinfluenceofADPNontheexpressionofthecellcycleregulatorsandapoptosisgene，westernblottinganalyzedthephosphorylationofAMPKinducedbyADPNinHelacellsTheresultsshowedmatADPNreceptorsareexpressedinMGC一803、BGC一823、HT29、H

8、elacellsADPNWassensitivetotheHelacells，O1Itg／mlADPNCaninhibitthegrowthofHelacellsandlOItg／mlADPNinducedHelacellstoapoptosisADPNdownregulatedtheexpressionofcellpositiveregulatorscyclin—D1andcmyc，upregulatedtheexpressionof