糖原合成酶激酶-3β对巨噬细胞系RAW264.7活化的影响.pdf

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1、·610·ChineseHepatology，Aug．2014，Vo1．19，No．8·论著·糖原合成酶激酶一3I3对巨噬细胞系RAW264．7活化的影响张丹丹李珊珊李璐李俊峰郑素军段钟平陈煜【摘要】目的探讨糖原合成酶激酶一3p(GSK一3p)对巨噬细胞系RAW264．7活化的影响。方法将生长状态良好的RAW264．7细胞分为3组：实验对照组、脂多糖(LPS)组和GSK一3日特异抑制剂SB216763干预组，在12h、24h进行指标检测。采用Western印迹法检测细胞GSK一3p、p-GSK一3p蛋白的表达，ELISA

2、试剂盒法检测细胞上清IL一1()、TNF_的变化，RT—PCR检测细胞中5-LOmRNA变化，免疫荧光法检测ED1表达，电镜下观察RAW264．7细胞形态变化。结果12h和24hLPS组与对照组相比，p-GSK一3p表达减少，GSK一3口表达不变，活性升高；TNF—a、IL一10及5一I0mRNA表达增多(P<0．05)；ED1表达绿色荧光明显增多；透射电镜观察LPS组巨噬细胞形态不规则，吞噬坏死物质细胞变多。12h和24hSB216763干预组与LPS组进行比较，p-GSK一3表达明显增多，GSK一3p表达不变，活性降

3、低；TNF—n及5一LOmRNA表达减少(P

4、，DUANZhong—ping，CHENYu．Artificiallivercenter，BeijingYouAnHospital，CapitalMedicalUniversity，Beijing100069，ChinaCorrsp0”gauthor：CHENYu，Email：chybeyond@163．corn[Abstract]ObjectiveToinvestigatetheeffectofglycogensynthasekinase一3口(GSK一38)ontheactivationofmacrophagecel

5、llineRAW264．7．MethodsRAW264．7cellswhichgrewwellweredividedintothreegroups：theexperimentalgroup，lipopolysaccharide(LPS)groupandGSK一3BspecificinhibitorSB216763interventiongroup．Indexesdetectionwasperformedat12and24hoursafterLPSorGSK一38specificinhibitorintervention．

6、TheexpressionofGSK一3Bandp-GSK一3~ser9proteinincellswasdetectedbyusingwesternblot，whileIL一10，sCD163andTNF-aincellsupernatantwereexaminedbyELISAassaykit．RT-PCRwasperformedtodetect5-LOmRNAincells，andimmunofluorescencewasperformedforED1expression．Inaddition，themorphol

7、ogyofRAW264．7cellswereobservedbyelectronmicroscopy．ResultsComparedwithcontrolgroup，eachindexinLPSgroupat12hand24hshowedthat：P—GSK一313ser9expressionwasdecreased，GSK一3pexpressionremainedunchanged，however，activityofGSK一3pwasincreased；thereweresignificantlyincreasedi

8、nmRNAlevelsofTNF-a，sCD163，IL一10and5-LO(P<0．05)；expressionofED1withgreenfluorescencewasalsosignificantlyincreased；undertransmissionelectronmicroscopy(TEM)，themo