钠氢交换体-1敲降对缺氧巨噬细胞三磷酸腺苷结合盒转运体A1水平及胆固醇外排的影响  

Effects of Na^＋ -H^＋ exchanger 1 knockdown on protein expression levels of ATP binding cassette transporter A1 and cholesterol efflux in hypoxic RAW264.7 cells

作　　者：莫显刚[1] 张莉[1] 张落超 洪伟[2] 王兰[1] 代陆军[3] 文前军[4] 

机构地区：[1]贵州医科大学附属医院综合病房,贵阳550004 [2]贵州医科大学医学分子生物学贵州省重点实验室,贵阳550004 [3]贵州医科大学附属医院病理科,贵阳550004 [4]贵州医科大学附属医院感染科,贵阳550004

出　　处：《中华老年医学杂志》2017年第8期909-914,共6页Chinese Journal of Geriatrics

基　　金：国家自然科学基金（31260250、31660288）

摘　　要：目的探讨钠氢交换体-1（NHE1）基因敲降对缺氧状态下巨噬细胞三磷酸腺苷结合盒转运体A1（ABCA1）蛋白水平及胆固醇外流的影响。方法感染表达特异靶向NHE1基因短发夹RNA慢病毒（siNHE1）或表达乱码RNA慢病毒（siNC）的RAW264．7细胞缺氧24h，实时定量PCR（qRT-PCR）及免疫印迹（Western blot）检测NHE1 mRNA及蛋白表达水平，用sNARF-1、Fluo-4NW及Suc-LLVY-aminoluciferin分别检测NHE1活性、细胞内钙离子浓度（[Ca^2＋]，）及钙蛋白酶（calpain）活性；免疫印迹检测缺氧状态下ABCA1蛋白水平，高效液相色谱法检测细胞内胆固醇含量及^3H-胆固醇法检测胆固醇外流。结果缺氧相对于常氧上调NHE1 mRNA、蛋白水平表达及活性2．48倍、1．28倍和61．96％（P〈0．05），升高[Ca^2＋]．及calpain活性4．51倍和2．41倍（P〈0．05），而siNHE1降低缺氧细胞NHE1 mRNA、蛋白表达及活性84．95％、60．75％和66．44％（P〈0．05），降低[Ca^2＋]。及calpain活性59．23％和54．66％（P〈0．05）。缺氧使ABCA1蛋白水平降低61．67％（P〈0．05），siNHE1使缺氧细胞ABCA1蛋白水平升高56．52％。缺氧使细胞内总胆固醇及胆固醇酯含量增加74．57％和101．81％（均P〈0．05），而siNHE1降低总胆固醇及胆固醇酯34．24％及49．66％（均P〈0．05）。缺氧降低胆固醇外流34．79％，而缺氧条件下siNHE1使胆固醇外流率升高37．80％。结论NHE1可能通过[Ca^2＋]。／calpain通路在缺氧诱导ABCA1蛋白水平降低及胆固醇外流异常发挥重要作用。Objective To explore the effects of Na^＋-H^＋ exchanger I（NHE1） knockdown on ATP binding cassette transporter A1 （ABCA1） protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells. Methods The RAW264. 7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1 （siNHE1） or scramble RNA （siNC）. The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition. In the meantime, the methods of SNARF 1, Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity, intracellular Ca^2＋ （[Ca^2＋ ]i ） and calpain activity, respectively. Furthermore,ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells. In parallel,the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HI,PC and ^3H-cholesterol. Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds, 1.28 folds and 61.96% （ali P〈0. 05 ）, and increased[Ca^2＋]i and calpain activity by 4.51 folds and 2.41 folds （all P 〈 0.05）. Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95% ,60. 75% and 66.44% ,respectively （all P〈0. 05） and[Ca^2＋]i and calpain activity were reduced by 59.23% and 54.66% （P〈0.05）. Furthermore,the ABCA1 protein level was 61.67% lower in the hypoxic cells than in the normoxic cells （P〈0.05） ,and siNHE1 was increased by 56.52% after treatment of Hypoxia. Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57% and 101.81% （all P〈0. 05）. Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24%及 49.66% （all P〈0.05）. Hypoxia reduced the cholesterol efflux by 34.79% （P〈0. 05）, which were partially rever

关 键 词：三磷酸腺苷结合盒转运体A1 钠氢交换体-1 胆固醇 

分 类 号：R543.5[医药卫生—心血管疾病]