预览加载中，请您耐心等待几秒...

高糖环境对大鼠肾小球系膜细胞AMPK表达及活性的影响.docx

预览
1/4
2/4
3/4
4/4

在线预览结束，喜欢就下载吧，查找使用更方便

下载提示 文本预览

如果您无法下载资料，请参考说明：

1、部分资料下载需要金币，请确保您的账户上有足够的金币

2、已购买过的文档，再次下载不重复扣费

3、资料包下载后请先用软件解压，在使用对应软件打开

高糖环境对大鼠肾小球系膜细胞AMPK表达及活性的影响 Introduction Diabeticnephropathyisoneofthemostcommoncomplicationsassociatedwithdiabetesmellitus.Thepathologyofdiabeticnephropathyischaracterizedbychronicinflammation,oxidativestress,andintricatesignalingpathways.Somestudieshavesuggestedthathighglucoseenvironmentscanactivatesignalingpathways,leadingtotheexpressionofinflammatorycytokines,chemokines,andmatrixproteins.Activationofadenosinemonophosphate-activatedproteinkinase(AMPK)isknowntoregulatevariouscellularfunctions,anditsdysregulationmaycontributetothedevelopmentandprogressionofdiabeticnephropathy.Hence,thisstudyaimedtoinvestigatetheeffectofhighglucoseenvironmentsonAMPKexpressionandactivityinratmesangialcells. MaterialsandMethods AnimalModel Ten-week-oldSprague-Dawley(SD)ratswerepurchasedfromtheExperimentalAnimalCenterofCentralSouthUniversity(Changsha,China)andhousedinananimalfacilityat22°Cwitha12:12light-darkcycle.Theanimalswerefedwithstandardchowandwaterforaweekbeforetheexperiment. CellCulture Themesangialcellswereisolatedfromratkidneysaspreviouslydescribed.Briefly,thekidneysweredissectedandthecortexwascutintofinepieces.ThetissuesweredigestedwithcollagenaseandDNaseIbeforefilteringthecellsuspensionthrougha40-μmmesh.Thecellswereseededincultureflasks,maintainedinDulbecco'smodifiedEagle'smedium(DMEM)containing10%fetalbovineserum(FBS)and1%penicillinandstreptomycinandincubatedat37°Cwith5%CO2. GlucoseTreatment Themesangialcellswerestarvedfor24hoursinDMEMcontaininglow(5.5mM)glucoseandthentreatedwithhigh(25mM)glucosefordifferenttimeperiods(0,6,12,24,and48hours). CellViabilityAssay ThecellviabilitywasdeterminedusingtheMTTassay.Briefly,thecellswereseededin96-wellplatesandincubatedwithMTTsolutionfor4hours.Theformazancrystalsformedweredissolvedindimethylsulfoxideandtheabsorbancewasmeasuredat570nmusingamicroplatereader. Real-TimePCR ThetotalRNAwasextractedfromthemesangialcellsusingtheTRIzolreagentandreversetranscribedintocDNAusingthePrimeScriptTMReverseTranscriptionReagentKit.ThecDNAwasthenamplified