一磷酸鞘氨醇／一磷酸鞘氨醇1受体信号通路在大鼠肥大心肌细胞缺血后适应中的作用及其机制  

Role and related mechanism of S1P/S1P1 signal pathway during post conditioning of hypertrophic cardiomyocytes

作　　者：包馨慧 李海霞[1] 陶静[1] 李晓梅[1] 杨毅宁[1] 马依彤[1] 陈邦党[1] 

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054

出　　处：《中华心血管病杂志》2016年第5期431-435,共5页Chinese Journal of Cardiology

基　　金：国家自然科学基金（81060021）

摘　　要：目的探讨一磷酸鞘氨醇（S1P）／一磷酸鞘氨醇1型受体（S1P1）信号通路在肥大心肌细胞缺血后适应中的作用及其机制。方法将原代分离培养的SD乳鼠心肌细胞用去甲肾上腺素（NE）1μmol／L诱导肥大后，用三气培养箱缺氧复氧模拟缺血环境建立缺氧后适应模型并对其进行鉴定。诱导成功的肥大心肌细胞在缺氧后适应基础上添加药物干预，根据添加药物分为5组，即缺氧后适应组（IPost组，肥大心肌细胞建立缺氧血后适应模型，无其他特殊干预），缺氧后适应加S1P组（IPost＋S1P组，肥大心肌细胞在建立缺氧后适应模型前，先用S1P1μmol／L孵育2h），缺氧后适应加S1P1抑制剂W-146和slP组（IPost＋S1P＋W-146组，肥大心肌细胞在建立缺氧后适应模型前，先用W-1460．4μmo]／L处理20min然后添加SIP1μmol／L孵育2h），缺氧后适应加丝裂原活化蛋白激酶（MAPK）阻滞剂PD98059和S1P组（IPost＋SIP＋PD98059组，肥大心肌细胞在建立缺氧后适应模型前，先用PD98059125μmol／L处理20min后再添加s1P1μmol／L孵育2h），以及缺氧后适应加P13K阻滞剂LY294002和S1P组（IPost＋SIP＋LY294002组，肥大心肌细胞在建立缺氧后适应模型前，先用LY2940020．1μmol／L处理20rain然后添加S1P1μmol／L孵育2h）。采用流式细胞仪检测各组肥大心肌细胞的凋亡率。蛋白印迹法检测凋亡蛋白caspase-3及相关信号通路蛋白细胞外信号调节激酶1／2总蛋白（t-ERKI／2）、细胞外信号调节激酶1／2磷酸化蛋白（p-ERKI／2）、蛋白激酶B总蛋白（t-Akt）、蛋白激酶B磷酸化蛋白（p-Akt）的表达水平。结果（1）肥大心肌细胞缺氧后适应模型建立的鉴定结果：缺氧复氧、缺氧后适应培养的肥大心肌细胞以及正常培养的肥大心肌细胞凋亡率分别为（27．90±4．49）％、（15．90±1．77）％以及（7．97±2．18）％，前两者均明显高于正常培养的肥大Objective To study the role and mechanism of sphingosine-1-phosphate （SIP）/ sphingosine-l-phosphate receptor 1 （S1P1） signal pathway during post conditioning of hypertrophic cardiomyocytes. Methods Neonatal rat eardiomyocytes were isolated and cultured, then stimulated by norepinephrine （NE） to induce cardiomyocytes hypertrophy. Using tri-gas incubator to create hypoxia and reoxygenation enviroment to mimic ischemia-reperfusion and postconditioning. Hypertrophic cardiomyoctyes were divided into five groups according to the presence of absence of various drugs and postconditiong and relevant signal pathways changes were detected ： （ 1 ） IPost group （ hypoxia ＋ postconditioning） ; （ 2 ） IPost ＋ SIP group （cells were pretreated with SIP （1 μmol/L） for 2 h before IPost） ; （3） IPost ＋ W-146 ＋ S1P group （cells in IPost ＋ W-146 ＋ SIP group were pretreated with S1P1 inhibitor W-146 （0.4 μmol/L） for 20 min） ; （4） IPost ＋ PD98059 ＋ SIP group （cells in IPost ＋ SIP group were pretreated with MAPK antagonist PD98059 （125 μmol/L） for 20 min） ; （5） IPost ＋ LY-294002 ＋ SIP group （cells in IPost ＋ SIP group were pretreated with PI3K antagonist LY294002 （0. 1 μmol/L） for 20 rain）. Apoptosis was detected by flow cytometry and protein expression of relevant signal pathways were detected by Western blot. Results （ 1 ） Apoptosis rate was significantly increased in hypoxia/reoxygenation （ 27.90± 4. 49） % group compared with normal control group （ 7.97 ± 2. 18 ） % , which could be significantly reduced in IPost group （ 15.90 ± 1.77 ） % （ all P 〈 0. 05 ）. （ 2 ） Apoptosis rate and caspase-3 expression were both significantly lower in IPost ＋ S1P and IPost ＋ S1P ＋ LY-294002 groups than in IPost and IPost ＋ S1P ＋ W-146 and IPost ＋ SIP ＋ PD98059 group （all P 〈 0. 05 ）. （3）p-ERK1/2 expression was significantly higher in IPost ＋ S1P and IPost ＋ S1P ＋ LY-294002 group than in IPost and IPost

关 键 词：心肌再灌注损伤 受体 鞘磷脂 细胞凋亡 

分 类 号：R54[医药卫生—心血管疾病]