高密度脂蛋白拮抗氧化型低密度脂蛋白诱导的血管平滑肌细胞中胱硫醚γ裂解酶表达的下调

发布时间：2018-01-01 00:27

本文关键词：高密度脂蛋白拮抗氧化型低密度脂蛋白诱导的血管平滑肌细胞中胱硫醚γ裂解酶表达的下调　出处：《福建医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:高密度脂蛋白(High density lipoprotein,HDL)拮抗氧化型低密度脂蛋白(Oxidized low density lipoprotein,ox-LDL)诱导的氧化应激在保护血管内皮细胞和平滑肌细胞中发挥重要作用。本实验探究了ox-LDL和HDL对血管平滑肌细胞(Vascular smooth muscle cell,VSMC)中气体信号分子硫化氢(Hydrogen sulfide,H2S)催化酶胱硫醚γ-裂解酶(Cystathionine gamma-lyase,CSE)表达的影响,以及HDL能否通过拮抗氧化应激来抑制ox-LDL对于CSE表达的调节作用及其中参与的机制。方法:大鼠主动脉血管平滑肌细胞在无菌环境下分离培养。从健康志愿者中提取的血浆经超速离心分离HDL和LDL,ox-LDL由硫酸铜(Cu SO4)氧化而得。CSE、血红素加氧酶1(heme oxygenase 1,HO-1)蛋白表达和ERK1/2、AKT、p38/MAPK的磷酸激活利用免疫印迹法检测,CSE m RNA表达用实时荧光定量PCR法测定。细胞内活性氧(Reactive oxygen species,ROS)释放用ROS介导的活性氧检测探针(DCFH-DA)检测。用亚甲基蓝显色测吸光度法测定H2S生成。RNAi技术沉默VSMC中HO-1的表达。结果:Ox-LDL能够呈时间和浓度依赖性地下调VSMC中CSE蛋白和m RNA表达。HDL不仅能直接促进VSMC中CSE蛋白表达和H2S释放,还能拮抗ox-LDL对CSE的下调作用。HDL能诱导HO-1生成并拮抗由ox-LDL诱导的胞内ROS释放。HDL诱导ERK、AKT、p38/MAPK磷酸化,ERK1/2、AKT、和p38/MAPK抑制剂能够减弱HDL诱导的HO-1表达并削弱对ox-LDL诱导的ROS释放的清除能力。ERK1/2、AKT、和p38/MAPK抑制剂能够削弱HDL诱导的CSE蛋白表达及HDL对ox-LDL诱导的CSE表达的拮抗作用。干扰VSMC中HO-1的表达,HDL对CSE表达的诱导作用减弱。结论:HDL能通过诱导HO-1表达拮抗ox-LDL诱导的VSMC中ROS释放和CSE表达下调,HDL还可直接诱导CSE蛋白表达和H2S的释放,且ERK1/2、AKT、p38MAPK信号通路参与其中。
[Abstract]:Objective: to study the high density lipoprotein of high density lipoprotein. Hd) antagonized oxidized low density lipoprotein (low density lipoprotein). Oxidative stress induced by ox-LDL plays an important role in the protection of vascular endothelial cells and smooth muscle cells. In this study, we investigated the effects of ox-LDL and HDL on vascular smooth muscle cells (VSMC). Vascular smooth muscle cell. Gas signal molecule hydrogen sulfide Hydrogen sulfide. The effect of cystathionine gamma-lyase (CSE) on the expression of cystathionine 纬 -lyase catalyzed by H _ 2S was observed. And whether HDL can inhibit the regulatory effect of ox-LDL on the expression of CSE and its mechanism by antagonizing oxidative stress. Methods:. Rat aortic vascular smooth muscle cells were isolated and cultured in aseptic environment. HDL and LDL were isolated from plasma from healthy volunteers by ultracentrifugation. Ox-LDL was oxidized by CuSO4) to obtain. CSE, heme oxygenase 1 (HO-1) protein expression and ERK1/2. The phosphoric acid activation of AKT p38 / MAPK was detected by Western blotting. The expression of CSE m RNA was assayed by real-time fluorescence quantitative PCR. Intracellular reactive oxygen species was detected by reactive oxygen species. Ros) release using ROS mediated active oxygen species detection probe DCFH-DAA. Detection of HO-1 in VSMC by methylene blue colorimetric assay. Results:. Ox-LDL can down-regulate the expression of CSE protein and m RNA in VSMC in a time-and concentration-dependent manner. HDL-C can not only directly promote the expression of CSE protein and H2S release in VSMC. It could also antagonize the down-regulation of CSE by ox-LDL. HDL could induce HO-1 production and antagonize the intracellular ROS release induced by ox-LDL. HDL-induced ERKK AKT. P38 / MAPK phosphorylated ERK1 / 2 AKT. P38 / MAPK inhibitor could attenuate the expression of HO-1 induced by HDL and weaken the clearance of ROS release induced by ox-LDL. P38 / MAPK inhibitor could weaken the expression of CSE protein induced by HDL and the antagonism of HDL to CSE expression induced by ox-LDL, and interfere with the expression of HO-1 in VSMC. Conclusion HDL can antagonize the release of ROS and the down-regulation of CSE expression in VSMC induced by ox-LDL by inducing HO-1 expression. HDL can also directly induce the expression of CSE protein and the release of H2S, and ERK1 / 2 AK p38 MAPK signaling pathway is involved.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R541.75

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