rnai抑制大鼠睾丸支持细胞upa基因表达的分析

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1、华中科技大学硕士学文论文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华中科技大学硕士学文论文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