生理学报ActaPhysiologicaSinica,August25,2008,60(4):511-519http://www.actaps.com.cn511研究论文Received2008-03-03Accepted2008-04-03ThisworkwassupportedbytheNaturalScienceFoundationofHunanProvince,China(No.05JJ40039)andNationalInstitutesofHealth,USA(No.HL-57352).*Correspondingauthor.Tel:+1-919-966-9286,Fax:+1-919-843-5945,E-mail:mrunge@med.unc.eduNADPH氧化酶活性不影响主动脉平滑肌细胞负荷胆固醇袁中华1，NageswaraR.Madamanchi2，AleksandrE.Vendrov2，NIUXi-Lin2，LIJu-Xiang2，MarschallS.Runge2,*1南华大学医学院心血管病研究所，动脉硬化学湖南省重点实验室，衡阳421001；2美国北卡罗来纳大学医学院卡罗来纳心血管生物学中心，教堂山27599摘要：NADPH氧化酶产生的活性氧促进血管平滑肌细胞的增殖和迁移，与动脉粥样硬化的发生密切相关。为了观察NADPH氧化酶的亚基p47phox对血管平滑肌细胞胆固醇代谢的影响，把p47phox基因敲除小鼠的主动脉血管平滑肌细胞与10mg/L水溶性胆固醇共孵育72h，然后用0.3mg/L凝血酶处理10min，采用免疫组织化学和油红O染色、实时定量逆转录PCR、免疫蛋白印迹、细胞内胆固醇测定等方法，观察细胞内胆固醇的改变，与平滑肌细胞、巨噬细胞、炎症反应细胞内胆固醇代谢相关蛋白的表达。结果显示，与未孵育的对照组相比，水溶性胆固醇孵育过的主动脉血管平滑肌细胞内胆固醇明显增加，差别有显著性意义；细胞内中性脂滴明显增加；α-肌动蛋白的表达下降，半乳糖凝集素3表达升高，单核细胞趋化蛋白1及血管细胞黏附分子1的表达不变；ATP结合盒转运体A1、酰基辅酶A：胆固醇酰基转移酶1及脂肪分化相关蛋白的表达增加。但是，与野生型血管平滑肌细胞相比，敲除p47phox基因并不能使所测定的指标发生变化。结果提示，负荷胆固醇后，p47phox依赖的NADPH氧化酶并不能改变血管平滑肌细胞向泡沫细胞的转变。单纯敲除p47phox基因不能改变细胞内胆固醇代谢的状态。关键词：NADPH氧化酶；胆固醇；基因敲除；平滑肌细胞；动脉粥样硬化中图分类号：R363NADPHoxidaseactivitydoesnotaffectcellularcholesterolloadinginvascularsmoothmusclecellsYUANZhong-Hua1,NageswaraR.Madamanchi2,AleksandrE.Vendrov2,NIUXi-Lin2,LIJu-Xiang2,MarschallS.Runge2,*1InstituteofCardiovascularDisease,KeyLaboratoryforArteriosclerologyofHunanProvince,UniversityofSouthChina,Hengyang421001,China;2CarolinaCardiovascularBiologyCenter,DepartmentofMedicine,UniversityofNorthCarolina,ChapelHill,NC27599,USAAbstract:ReactiveoxygenspeciesgeneratedbyNADPHoxidaseenhanceaorticvascularsmoothmusclecellproliferationandmigra-tionwhichplayanimportantroleinthepathophysiologyofatherosclerosis.WeinvestigatedtheroleofNADPHoxidaseinthecellularcholesterolmetabolisminvascularsmoothmusclecellsusingp47phox-deficientcells.Wild-typeandp47phoxknockoutvascularsmoothmusclecellswereloadedwithcholesterolfor72hbyusing10mg/Lcholesterol:methyl-β-cyclodextrincomplexesandthenincubatedwithorwithout0.3mg/Lthrombinfor10min.Foamcellformationwasdeterminedbyaccumulationofintracellularcholesterol,oilRedO-stainedlipiddroplets.Aftercholesterolloading,cellularlipiddropletsraisedsharply,cellularcholesterolincreasedfrom(31.4±2.0)to(61.0±2.1)mg/gprotein(P<0.05)inwild-typecells,andfrom(29.8±2.5)to(51.3±3.1)mg/gprotein(P<0.05)inp47phoxdeficientcells,butthedifferencebetweenthetwocelltypeswasnotsignificant.Immunostainingshoweddecreasedlevelsofsmoothmuscleα-actinandincreasedlevelsofmacrophagemarkerMac-2inbothwild-typeandp47phoxdeficientvascularsmoothmusclecells.Oneofthemacrophage-relatedinflammationgenes,monocytechemoattractantprotein-1(MCP-1)expressiondid生理学报ActaPhysiologicaSinica,August25,2008,60(4):511-519512notchangeinbothtwocelltypesdetectedbyimmunostaining.Although...
