干扰srebp-1c对内质网应激状态下l02和hepg2肝细胞脂质沉积的影响

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1、干扰SREBP-1c对内质网应激状态下L02和HepG2肝细胞脂质沉积的影响房殿亮，宁波，沈薇，万颖（400010重庆，重庆医科大学附属第二医院消化内科）[摘要]目的构建SREBP-1c基因的miRNA真核表达载体，观察其对内质网应激状态下L02和HepG2肝细胞脂质沉积的影响。方法设计并构建SREBP-1c基因的3个miRNA干扰载体，经测序鉴定miRNA载体构建成功后转染肝细胞，荧光显微镜下观察绿色荧光蛋白表达。Westernblot检测转染后SREBP-1c的表达；甘油三酯测定和油红O染色检测细胞内脂质沉积；Westernblot检测

2、SREBP-1c下游脂代谢相关基因脂肪酸合成酶（fattyacidsynthase,FAS）和乙酰辅酶A羧化酶(acetylcoAcarboxylase,ACC1)的表达。结果靶向干扰SREBP-1c的miRNA真核表达载体构建成功。Westernblot结果显示，转染SREBP-1c-1miRNA和SREBP-1c-3miRNA载体后，L02和HepG2细胞内SREBP-1c蛋白水平表达均明显减低（P﹤0.05），SREBP-1c-1miRNA的干扰效果最好（P﹤0.05）。与对照组相比，转染SREBP-1c-1miRNA载体后L02和H

3、epG2肝细胞内甘油三酯含量均明显降低（P﹤0.05），细胞内脂滴明显减少；FAS和ACC1蛋白的表达明显降低（P﹤0.05）。结论SREBP-1c基因沉默通过FAS/ACC1降低了内质网应激状态下肝细胞脂质合成。[关键词]非酒精性脂肪肝；SREBP-1c；FAS；内质网应激；RNA干扰[中图法分类号]R575.5;R589.2;R363.2[文献标志码]ATheeffectofSREBP-1cgeneknockdownonlipidaccumulationinL02andHepG2hepatocytesunderendoplasmicre

4、ticulumstressFangDianliang,NingBo,ShenWei,WanYing(DepartmentofGastroenterology,SecondAffiliatedHospitalofChongqingMedicalUniversity,Chongqing,400010China)[Abstract]ObjectiveToconstructmiRNAeukaryoticexpressionvectorstargetingSREBP-1candtoobservetheeffectofSREBP-1cgeneknock

5、downonlipidaccumulationinL02andHepG2hepatocytesunderendoplasmicreticulumstress.MethodsThreemiRNAtargetingSREBP-1cgene,namedSREBP-1c-1-miRNA,SREBP-1c-2-miRNA,SREBP-1c-3-miRNAandnon-homologousnegativecontrolexpressionvector(HK-miRNA)weredesignedandconstructedwhichwereidentif

6、iedbyDNAsequencinganalysis.ThreemiRNAexpressionvectorsweretransientlytransfectedintohepatocytesandEGFPwasobservedunderfluorescencemicroscope.Tofilteroutthemostefficientinterferenceplasmid,theSREBP-1cexpressionwasdeterminedbyWesternblot.Lipiddepositionineachgroupwasobserved

7、bytriglyceridecontentdetectionandoilredOstaining.Westernblotwasappliedtomeasurefattyacidsynthase(FAS)andAcetylCoAcarboxylase(ACC1)proteinlevel.ResultsFourmiRNAexpressionvectorswereconstructedsuccessfully.After24htransfection,EGFPexpressionwasvisiableunderfluorescencemicros

8、cope.ThelevelofSREBP-1cproteinexpressiondecreasedsignificantlyaftertransfectedwithSREBP-1