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时间:2020-05-18
《糖原合成酶激酶-3β对巨噬细胞系RAW264.7活化的影响.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
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(P4、,DUANZhong—ping,CHENYu.Artificiallivercenter,BeijingYouAnHospital,CapitalMedicalUniversity,Beijing100069,ChinaCorrsp0”gauthor:CHENYu,Email:chybeyond@163.corn[Abstract]ObjectiveToinvestigatetheeffectofglycogensynthasekinase一3口(GSK一38)ontheactivationofmacrophagecel5、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,themorphol7、ogyofRAW264.7cellswereobservedbyelectronmicroscopy.ResultsComparedwithcontrolgroup,eachindexinLPSgroupat12hand24hshowedthat:P—GSK一313ser9expressionwasdecreased,GSK一3pexpressionremainedunchanged,however,activityofGSK一3pwasincreased;thereweresignificantlyincreasedi8、nmRNAlevelsofTNF-a,sCD163,IL一10and5-LO(P<0.05);expressionofED1withgreenfluorescencewasalsosignificantlyincreased;undertransmissionelectronmicroscopy(TEM),themo
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
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