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1、华中科技大学硕士学位论文PartⅡThedifferencesofpulmonaryartery,femoralarteryandfemoralveinsmoothmusclecellsinmitochondrianumberandactivityObjectives：(1)Comparingtheamountofmitochondrialocatedinpulmonaryartery,femoralartery,femoralveinsmoothmusclecells.(2)Thecontentofcitratesynthase(CS)weredetectedb
2、yElisatoshowedtheamountofmitochondriaindirectly(3)Torevelthemitochondiralactivityofpulmonaryartery,femoralarteryandfemoralveinsmoothmusclecellsbydetectingmitochondrialsuccinatedehydrogenase(SDH)activity.Methods：1.mitochondrialisolation:SDmaleratsweighting150gwerekilledbycervicaldisloc
3、ation，andthenisolatedpulmonaryartery，femoralartery，femoralveininicebathofD-hank’ssolution.Removetheadventitiaandtheintimaundermicroscopetoobtainsmoothmuscle.Smoothmuscleswereplacedinice-coldisolationbuffer,andthetissuewasmincedwithscissorsandhomogenizedinicewithaglasshomogenizer.Aprot
4、easeinhibitorcocktail(20μL/2mLhomogenate;P8340;Sigma,St.Louis,MO)wasaddedtothehomogenateswithtrypsin.Thehomogenateswerecentrifugedat600gfor5minutesat4℃,andthesupernatantwasdecantedandcentrifugedat5000gfor10minutesat4℃.Theresultantmitochondrialpelletswereresuspendedinisolationbufferand
5、werestoredoniceuntilsubsequentuse.Methodchanges:centrifugalforcegradientchangeto1000g,10min/10000g,15minor600g,5min/7000g,10min.2.Mitochondriacount:mitochondriaresuspendedin100μLMito-track-greendyeat37℃for30mins,thencentrifugedin7000gat4℃for10mins,resuspendedtheprecipitationinhomogena
6、tebufferafterremovingsupernatant,Centrifugedin7000gat4℃for15minaftervortex,thenadded10μLofthestainedmitochondriasuspensiontocellcountboard,obtainedtheConcentrationofmitochondriasuspensionbycountingbrightfluorescencewithfluorescencemicroscope.3.Proteinconcentrationassay:Determineprotei
7、ncontentusing40μLsupernatantobtainedafterthefristcentrifugation.Mitochondrianumber/proteinquality=Mitochondrianumberpermassunit.4.Citratesynthasecontentassay:divided1mLofthesupernatantafterthefirstcentrifugationintofive,thenstoredat-20℃,determinedCScontentinaccordancewiththeELISAkitin
8、struc
