同型半胱氨酸硫内酯诱导半胱氨酸天冬氨酸蛋白酶12途径对内皮细胞凋亡的影响  

Effect of homocysteine thiolactone-induced Caspase-12 pathway on apoptosis of ECV-304 cells

作　　者：孟天娇 韩磊 姚尚争 刘影 孙文萍 Meng Tianjiao;Han Lei;Yao Shangzheng;Liu Ying;Sun Wenping(Department of Neurology,Zhejiang Zhuji People's Hospital,Zhuji 311800,Zhejiang Province,China)

机构地区：[1]浙江省诸暨市人民医院神经内科,311800 [2]大连金州新区人民医院神经内科 [3]北华大学附属医院神经内科 [4]上海市松江区中心医院全科医学科

出　　处：《中华老年心脑血管病杂志》2019年第12期1314-1317,共4页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：吉林省自然科学基金（20180101144JC）;吉林省教育厅“十三五”科学计划项目（JJKH20170063KJ）;吉林省卫生计生委科技能力提升计划（2017J079）

摘　　要：目的探讨同型半胱氨酸硫内酯(HTL)是否通过半胱氨酸天冬氨酸蛋白酶(Caspase)12凋亡途径影响内皮细胞生长。方法体外培养内皮细胞ECV304,先将0、50、100和200μmol/L的HTL干预细胞24 h,MTT法检测细胞增殖抑制率;随后以200μmol/L的HTL干预0 h、3 h、16 h和24 h,流式细胞术检测细胞周期和细胞凋亡情况,再以200μmol/L HTL刺激细胞为观察组(3、16和24 h),不加HTL刺激的细胞为对照组,2组分别采用免疫组织化学染色和Western Blot法检测Caspase-12、Caspase-3和Bcl-2蛋白表达情况。结果100和200μmol/L的HTL作用细胞24 h,细胞活性分别为0.76±0.01、0.73±0.01;200μmol/L HTL对细胞的抑制作用具有时间依赖性,与0 h比较,3、16和24 h的细胞活性下降明显(0.87±0.04、0.83±0.04、0.78±0.01 vs 0.90±0.07,P<0.01)。与对照组比较,观察组Caspase-12蛋白、Caspase-3蛋白表达增加(P<0.05,P<0.01);Bcl-2蛋白先增高后降低(P<0.01)。结论Caspase-12途径参与HTL致细胞凋亡的过程,Bcl-2也参与其调控。Objective To study whether HTL can influence the growth of endothelial cells ECV304 through the Caspase12 apoptosis pathway.Methods ECV304 were cultured in vitro and preconditioned at the concentration of 50,100 and 200μmol/L HTL respectively.The inhibition rate of ECV304 proliferation was detected with MTT staining.After the ECV304 were reconditioned in 200μmol/L HTL for 0,3,16 and 24 h respectively,the cell cycle and apoptosis of ECV304 were detected by flow cytometry.The ECV304 stimulated in 200μmol/L HTL served as an observation group(for 3,16 and 24 h)and those stimulated without HTL served as a control group.The expressions of Caspase-3,Caspase-12 and Bcl-2 ptoteins were detected by Western bolt with immunohistochemical staining.Results The activity of ECV304 stimulated in 100μmol/L HTL and 200μmol/L HTL was significantly higher in observation group than in control group(0.76±0.01 vs 0.73±0.01,P<0.05).The inhibitory effect of 200μmol/L HTL was time-dependent.The activity of ECV304 stimulated for 0 h was significantly lower than that of ECV304 stimulated for 3,16 and 24 h(0.87±0.04,0.83±0.04,0.78±0.01 vs 0.90±0.07,P<0.01).The expression levels of Caspase-12 and Caspase-3 increased(P<0.01)while that of Bcl-2 increased at first and then decreased with the prolongation of stimulation time(P<0.01).Conclusion The Caspase-12 and Bcl-2 apoptosis pathway is involved in HTL-induced apoptosis of ECV304.

关 键 词：高半胱氨酸 半胱氨酸天冬氨酸蛋白酶12 内皮细胞 细胞周期 细胞凋亡 流式细胞术 

分 类 号：R73[医药卫生—肿瘤]