血红素氧合酶-1在空气细颗粒物PM2.5致人脐静脉内皮细胞损伤过程中作用的实验研究  

Involvement of heme oxygenase in PM2.5-toxicity in human umbilical vein endothelial cells

作　　者：杨竞路 吕吉元[1] 张明升[2] 秦纲[1] 李彩萍[1] 

机构地区：[1]山西医科大学第一附属医院心内科,太原030001 [2]山西医科大学第一附属医院药理学教研室,太原030001

出　　处：《中华心血管病杂志》2013年第11期955-961,共7页Chinese Journal of Cardiology

基　　金：国家自然科学基金资助项目（81070225）;山西省国际科技合作项目（2012081046）

摘　　要：目的 通过观察活性氧（reactive oxygen species,ROS）清除剂（钛试剂,Trion）和血红素氧合酶-1 （heme oxygenase,HO-1）抑制剂（锌原卟啉,ZnPP）对空气细颗粒物PM2.5所致人脐静脉内皮细胞（HUVEC）的生物作用的影响,探讨HO-1在PM2.5致细胞损伤中的作用机制.方法 复苏由上海拜力生物科技有限公司提供的人脐静脉内皮细胞株,37℃、5％ CO2条件下正常培养至细胞形成致密单层时,分别进行干预.实验分组：（1）空白对照组（对照组）：细胞正常培养24 h.（2）PM2.5染毒组（PM2.5组）：不同浓度PM2.5（200、400、800 μg/ml）培养细胞24 h,选择400 μg/ml为其有效干预浓度（后续各组如未特殊注明,默认PM2.5浓度为400 μg/ml）.（3）PM2.5＋Trion组：ROS抑制剂Trion（10μmol/L）干预细胞1h后,加入PM2.5,培养24 h.（4）PM2.5＋ZnPP组：HO-1抑制剂ZnPP（10μmol/L）干预细胞1h后,加入PM2.5,培养24 h.收集干预后的各组细胞,用MTT法比较细胞活性,逆转录聚合酶链反应法和免疫细胞荧光法分析细胞内HO-1 mRNA及蛋白表达情况,荧光探针标记法测定细胞内ROS水平,流式细胞技术测定细胞凋亡率,分光光度法测定caspase-3活性.结果 PM2.5组细胞死亡率和凋亡率均高于对照组（P均＜0.05）.PM2.5＋Tiron组细胞死亡率和凋亡率均低于PM2.5组,PM2.5＋ ZnPP组则均高于PM2.5组（P均＜0.05）.PM2.5组ROS水平高于对照组（P＜0.05）.PM2.5＋Tiron组ROS水平低于PM2.5组,PM2.5＋ZnPP组则高于PM2.5组（P均＜0.05）.PM2.5组HO-1 mRNA和蛋白表达均高于对照组（P分别＜0.05和0.01）.PM2.5＋Tiron组HO-1 mRNA和蛋白表达均低于PM2.5组,PM2.5＋ZnPP组亦均低于PM2.5组（P＜0.05或0.01）.3、6、12和24 h不同时间干预后,PM2.5组细胞caspase-3活性均高于对照组（P均＜0.05）,PM2.5＋Tiron组均低于PM2.5组（P均＜0.05）,PM2.5＋ ZnPP组则均高于PM2.5组（P均＜0.05）.结论 PM2.5可以通过增加ROS损害HUVEC,同时引起HOObjective To investigate the involvement of heme oxygenase （HO-1） in PM2. 5 induced toxic responses in human umbilical vein endothelial cells （HUVECs）. Methods The experiment groups are as follows： （1） control group; （2） PM2. 5 groups： the cells were cultured with various concentrations of PM2. 5 （200, 400, 800μg/ml） for 24 h and 400 μg/ml was chosen for the main study; （ 3 ） PM2.5 ± Trion group ： the cells were pre-treated by 10 μmo//L Trion ~ a scavenger of reactive oxygen species（ROS） ] for 1 h before PM2. 5 （400 μg/ml） treatment for 24 h; （4） PM2. 5 ± ZnPP group： the cells were pretreated by HO-1 inhibitor ZnPP （10μmol/L） for 1 h before treatment with PM2. 5 （400wg/ml） for 24 h. MTT assay was used to detect cell viability. Reverse transcription polymerase chain reaction （RT-PCR） and indirect immunofluorescence assay were used to determine the mRNA and protein expressions of HO-1. Fluorescence labeling probe method was used to measure intracellular ROS level and flow cytometry was used for cell apoptosis. Colorimetric assay was used to detect intracellular caspase-3 activity. Results Compared with control, PM2. 5 significantly decreased celi viability, increased intracellular ROS, cell apoptosis and caspase-3 activity （ all P 〈 0.05 ）, these effects were significantly attenuated in PM2. 5 ± Tiron group while enhanced in PM2. 5 ± ZnPP group （ all P 〈 0. 05 vs. PM2. 5 group）. PM2. 5 upregulated HO-1 mRNA and protein expressions in HUVECs which was downregulated in both PM2. 5 ± Tiron group and PM2. 5 ± ZnPP group. Conclusion PM2. 5 could induce oxidative injury through increasing ROS production via modulating HO-1 mRNA and protein expressions, the injury could be aggravated with inhibition of the activity of HO-1 suggesting a potential protective role of HO-1 against PM2. 5 induced oxidative stress in HUVECs.

关 键 词：内皮细胞 活性氧 血红素加氧酶-1 PM2 5 

分 类 号：R363[医药卫生—病理学]