载脂蛋白A-Ⅰ模拟肽对RAW264.7巨噬细胞胆固醇流出的影响  

Effect of apolipoprotein A-Ⅰ mimetic peptides on cholesterol efflux in RAW264.7 cells

作　　者：谢琼[1,2] 赵水平[1] 

机构地区：[1]中南大学湘雅二医院心内科,长沙410011 [2]湖南省人民医院心内科,长沙410005

出　　处：《中南大学学报（医学版）》2011年第1期51-57,共7页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(30770857)~~

摘　　要：目的:观察载脂蛋白A-Ⅰ(apoA-Ⅰ)模拟肽D-4F对RAW264.7巨噬细胞胆固醇流出的影响,并探讨其机制。方法:巨噬细胞种植于24孔板,用1.85×107Bq/孔3H-胆固醇和含50μg/mL氧化型低密度脂蛋白(ox-LDL)共同孵育24h后,给予不同浓度的D-4F(0～100μg/mL)干预24h,收集细胞用液体闪烁计数法检测胆固醇流出。采用酶联免疫吸附试验(ELISA)测定细胞内环磷酸腺苷(cAMP)含量,采用实时荧光定量PCR及Western印迹检测三磷酸腺苷结合盒转运体A1(ABCA1)的mRNA及蛋白表达。结果:D-4F呈浓度依赖及时间依赖性促进巨噬细胞内胆固醇流出,增加细胞内cAMP水平,上调ABCA1mRNA和蛋白表达。8-Br-cAMP显著增加D-4F介导的胆固醇流出和ABCA1表达,而蛋白激酶A(PKA)抑制剂虽然对基础胆固醇流出及ABCA1表达几乎无作用,却可以抑制8-Br-cAMP对胆固醇流出和ABCA1表达的促进作用。结论:D-4F促进巨噬细胞胆固醇流出,cAMP释放及ABCA1表达,可能与激活cAMP-PKA-ABCA1通路有关。Objective To determine the effect and possible mechanism of an apolipoprotein（apo） A-Ⅰ mimetic peptide,D-4F,on cholesterol efflux in RAW264.7 macrophages.Methods RAW264.7 macrophages were incubated in the medium containing 8-bromo cAMP（8-Br-cAMP,0.5 mmol/L） and ox-LDL（50 μg/mL） for 24 h.Then various concentrations of D-4F（0-100 μg/mL） or H89（20 μmol/L,a protein kinase A inhibitor） were added for the purpose of interference.The intracellular cyclic AMP（cAMP） level was determined by enzyme-linked immunoabsobant assay（ELISA）.ATP binding cassette transporter A1（ABCA1） expression in the macrophages was quantitated by real-time PCR and Western blot.Results D-4F significantly increased the cholesterol efflux in both concentration and time-dependent manner accompanied by the increase in the intracellular cAMP level,ABCA1 mRNA and protein expression.The effect of D-4F on cholesterol efflux ABCA1 expression was enhanced by 8-Br-cAMP.Although H89 did not affect the basal cholesterol efflux and ABCA1 expression,it could attenuate the effect of 8-Br cAMP.Conclusion D-4F affects cholesterol efflux,cAMP level,and ABCA1 expression in macrophages,which is likely involved in the pathway of cAMP/PKA/ABCA1.

关 键 词：载脂蛋白A-I模拟肽 胆固醇流出 巨噬细胞 三磷酸腺苷结合盒转运体A1 环磷酸腺苷 

分 类 号：R346[医药卫生—基础医学]