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《rnai抑制大鼠睪丸支持細胞upa基因表達的分析》由會員上傳分享，免費在線閱讀，更多相關(guān)內(nèi)容在學術(shù)論文-天天文庫。
1、華中科技大學碩士學文論文AbstractPartⅠPrimarycellcultureandidentificationofratSertolicellsObjective:ToobtainhighlypureSertolicellsfromrat’stestesandidentifyculturedcellswithseveralmethods.Methods:Testeswereisolatedfrom18～22daysoldSDrats,andsubjectedtosequentialenzymatictreatments:firstwith0.25%t
2、rypsin,thenwith0.1%hyaluronidase,lastwith0.1%collagenase.Thecellsuspensionwasculturedinincubatorundertheconditionof32℃and5%CO2for48h.Thenculturedcellsweretreatedwith20mmol/LhypoosmoticTris-HClsolutionandculturedcontinuously.Afterincubationofoneweek,culturedcellswereidentifiedwiththe
3、Acridineorangefluorescencestaining,theFeulgenstainingandtheimmunohistochemicalmethodtestingFasL.Results:Over95%culturedcellswereSertolicells.MostofculturedcellsexpressedahighlevelofFasLandtheirstructuralfeatureswerethesameasSertolicellsidentifiedwithothermethods.Conclusion:Highlypur
4、esertolicellscanbeobstainedbythisexperimentalmethod.Thecellculturesystemwehaveestablishediscompetenttothefollowingexperiments.Keywords:rat;Sertolicell;culturePartⅡTheuPAgeneinhibitedbyRNAinterferenceinratSertolicellsExperimentoneOptimizationoftransfectionconditionsfortransfectingrat
5、SertolicellswithsiRNAIV華中科技大學碩士學文論文Objective:ToexaminethefeasibilityofchemicallysynthesizedsiRNAinblockingtheexogenousgeneexpressioninratSertolicellsandtooptimizethetransfectioncondition.Methods:ThetesteswereobtainedfromSDrats.TheSertolicellswereisolatedandculturedinvitro.Chemically
6、synthesizedsiRNAtargetingGAPDHgeneatvariousconcentrationswasthentransferredintotheprimarySertolicellsbyusingTMLipofectamine2000.ThechangesofGAPDHmRNAlevelinratSertolicellswereanalyzedbyRT-PCR.TheviabilityoftransfectedSertolicellswasdeterminedbyusingMTTassay.Results:Theconcentrationo
7、f150nmol/LwasoptimalforspecificallyblockingthegeneexpressionofGAPDH,whereasthecytotoxicitywasfurtherincreasedbyelevatingtheconcentrationofsiRNA,200nmol/LofsiRNAleadtocytotoxicityobviously.Conclusion:siRNAisabletotriggerRNAinterferenceinratSertolicells.Attheconcentrationof150nmol/L,i
8、tcanspecificallyand
