内皮素-1对大鼠血管平滑肌细胞一氧化氮及硫化氢体系的影响  

Influence of endothelin- 1 on nitric oxide and hydrogen sulfide pathways in vascular smooth muscle cells in rats

作　　者：田小雨[1] 张清友[1] 黄娅茜[1] 张达 汤新景 金红芳[1] 杜军保[1] 唐朝枢[3] 孙燕[1] Tian Xiaoyu;Zhang Qingyou;Huang Yaqian;Zhang Da;Tang Xinjing;Jin Hongfang;Du Junbao;Tang Chaoshu;Sun Yan(Department of Pediatrics,Peking University First Hospital,Beijing 100034,China（Tian XY,Zhang QY,Huang YQ,Zhang D Jin HF,Du JB,Sun Y;School of Pharmaceutical Science,Peking University Health Science Center,Beijing 100191,China（Tang X J;Department of Physiology and Pathophysiology,Peking University Health Science Center,Beijing 100191,China（Tang CS)

机构地区：[1]北京大学第一医院儿科,100034 [2]北京大学医学部药学院,100191 [3]北京大学生理与病理生理学系,100191

出　　处：《中华实用儿科临床杂志》2018年第13期1013-1017,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81500325）

摘　　要：目的探讨不同浓度内皮素-1（ET-1）对大鼠血管平滑肌细胞（A7r5细胞株）内源性一氧化氮（NO）及硫化氢（H2S）体系的影响。方法将A7r5细胞分为对照组和实验组，在实验组中分别加入浓度为10－8～10－6 mol/L的ET-1，对照组加入等体积的无菌磷酸盐缓冲液（PBS），48 h后分别应用NO和H2S探针原位检测A7r5细胞中NO及H2S水平，分别采用NO测定试剂盒和敏感硫电极法检测细胞上清中NO及H2S水平，应用Western blot法检测A7r5细胞中诱导型一氧化氮合酶（NOS2）、内皮型一氧化氮合酶（NOS3）、胱硫醚-γ-裂解酶（CSE）、胱硫醚-β-合成酶（CBS）及细胞增殖核抗原（PCNA）的表达。结果10－8、10－7及10－6 mol/L ET-1处理的A7r5细胞内NO水平的相对荧光强度（0.078±0.080、0.075±0.002、0.056±0.009）均低于对照组（0.094±0.061），差异有统计学意义（F＝15.248，P〈0.05）；细胞上清中NO水平[（1.391±0.134） μmol/L、（1.219±0.280） μmol/L、（1.116±0.181） μmol/L]均明显低于对照组[（2.131±0.484） μmol/L]，差异有统计学意义（F＝20.833，P〈0.01）；NOS2 （0.457±0.097、0.462±0.116、0.438±0.180）明显低于对照组（0.721±0.222），差异有统计学意义（F＝6.196，P〈0.05）；NOS3蛋白表达差异无统计学意义（F＝2.669，P〉0.05）。10－8、10－7、10－6 mol/L ET-1组A7r5细胞内H2S水平的相对荧光强度（0.063±0.002、0.056±0.008、0.042±0.009）低于对照组（0.082±0.006），差异有统计学意义（F＝16.297，P〈0.01）；细胞上清中H2S水平[（19.961±3.428） μmol/L、（17.516±5.144） μmol/L、（14.481±4.885） μmol/L]均明显低于对照组[（29.439±4.236） μmol/L]，差异有统计学意义（F＝12.518，P〈0.01）；CBS蛋白表达（0.359±0.096、0.270±0.038、0.174±0.051）均低于对照组（0.707±0.107），差异有统计学意义（F＝20.833，P〈0.01），CSE蛋白表达差异无统计学意义（F＝0ObjectiveTo explore the effect of different concentrations of endothelin-1 （ET-1） on the endogenous nitric oxide （NO） and hydrogen sulfide （H2S） pathways of vascular smooth muscle cells （A7r5 cell lines）in rats.MethodsA7r5 cell lines were divided into the control group and the experimental group.ET-1 at a concentration of 10-8 -10-6 mol/L was added into the experimental group, and as for the control group, the same volume of sterile phosphate buffered saline （PBS） buffer solution was added.The content of NO and H2S in A7r5 cell lines was detected by fluorescent NO probe and H2S probe after ET-1 stimulation for 48 h, respectively.The content of NO in the supernatant was measured by NO assay kit at 48 h of the incubation.The content of H2S in the supernatant was measured by polarographic H2S sensor at 48 h of the incubation.The expressions of inducible nitric oxide synthase （NOS2）, endothelial nitric oxide synthase （NOS3）, cystathionine-γ-lyase （CSE）, cystathionine-β-synthase （CBS） and proliferating cell nuclear antigen （PCNA） were detected by the Western blot method.ResultsThe relative fluorescence intensity of the content of NO in the A7r5 cell lines of ET-1 10-8, 10-7 and 10-6 mol/L groups （0.078±0.080, 0.075±0.002, 0.056±0.009） was markedly lower than that in the control group（0.094±0.061）, and the differences were statistically significant（F=15.248, P〈0.05）; Compared with the control group[（2.131±0.484） μmol/L], the content of NO in the supernatant of the experimental groups [（1.391±0.134） μmol/L, （1.219±0.280） μmol/L, （1.116±0.181） μmol/L]was significantly decreased, and the differences were statistically significant（F=20.833, P〈0.01）; NOS2 protein expression（0.457±0.097, 0.462±0.116, 0.438±0.180） was decreased markedly compared with that of the control group（0.721±0.222）, and the differences were statistically significant（F=6.196, P〈0.01）, but the expression of NOS3 showed no significant differences（

关 键 词：内皮素-1 一氧化氮 硫化氢 血管平滑肌细胞 大鼠 

分 类 号：R54[医药卫生—心血管疾病]