硫化氢对平滑肌细胞内吞氧化型低密度脂蛋白的影响

发布时间：2018-04-11 21:48

本文选题：硫化氢 + 炔丙基甘氨酸　；参考：《苏州大学》2012年硕士论文

【摘要】：目的：探讨外源性硫化氢对血管平滑肌细胞内吞氧化型低密度脂蛋白的影响，并对其分子学机制进行初步探讨，为动脉粥样硬化的防治提供新的理论依据。方法：采用组织贴块法从大鼠腹主动脉分离培养血管平滑肌细胞(vascular smoothmuscle cell，VSMC)。培养基选用含10％胎牛血清(FBS)的DMEM，培养条件为5％CO_2、37℃。培养3～5天有细胞爬出，7～9天可进行第一次传代。实验选用3～4代VSMC。根据不同检测方法可分为：①用于MTT检测细胞活性的细胞接种于96孔板，按5×104/mL细胞密度每孔200μl；②用于油红O染色、免疫细胞化学检测的细胞接种于放有小圆玻片的24孔板内，按1×105/ml细胞密度每孔1ml；③.用于免疫印迹检测的细胞接种于无菌6孔板中，按1×106/ml细胞密度，每孔2ml。实验分组： 1、MTT法检测H2S对VSMC活性影响：分为对照组、NaHS（25、50、100、200、500、1000μmol/L）组； 2、油红O染色检测VSMC脂质含量与免疫细胞化学法检测CD36、LOX-1受体表达：分为对照组、ox-LDL（80μg/ml）组、ox-LDL（80μg/ml）+NaHS(50μmol/L)组、ox-LDL（80μg/ml）+PPG(3mmol/L)组； 3、DiI-oxLDL内吞的检测：分为DiI-oxLDL（10μg/ml）组，DiI-oxLDL（10μg/ml）+NaHS(50μmol/L)组，DiI-oxLDL（10μg/ml）+PPG(3mmol/L)组； 4、Western blot测定VSMC的CD36、LOX-1蛋白表达：分为对照组、ox-LDL（80μg/ml）组、ox-LDL（80μg/ml）+NaHS（25、50、100、200μmol/L）组。结果： 1.MTT结果显示，NaHS浓度在200μmol/L以下时，对VSMC的活力没有明显影响，当NaHS浓度增加到500和1000μmol/L后细胞活力明显下降。 2.通过油红O染色观察外源性H2S（以NaHS为供体）对VSMC脂质摄取的影响。结果发现，与对照组相比，VSMC与ox-LDL单独孵育后胞内脂质显著增多，当用CSE（胱硫醚-γ-裂解酶）抑制剂PPG作用后，脂质沉积更明显，而H2S处理后的VSMC胞内脂质明显减少。 3.用DiI-oxLDL处理VSMC，Confocal观察VSMC对ox-LDL的内吞作用，主要观察外加DiI-oxLDL在VSMC胞内的积聚。结果观察到H2S能抑制VSMC对ox-LDL的内吞，而PPG则增强这种内吞作用，与油红O染色结果相符。 4.免疫细胞化学法检测VSMC的CD36、LOX-1受体表达，对H2S抑制VSMC内吞ox-LDL的分子学机制进行初步探讨。结果显示，ox-LDL能明显诱导VSMC的CD36、LOX-1受体表达，H2S处理后可下调这两种受体的表达，，PPG作用后则使CD36、LOX-1受体表达上调。 5.为进一步确定H2S抑制VSMC内吞脂质的机制是否与其下调CD36、LOX-1有关，本实验还采用了Western blot方法分析NaHS对VSMC的CD36、LOX-1蛋白表达的影响。结果发现，ox-LDL显著增强诱导VSMC的CD36、LOX-1蛋白表达，而H2S对两种受体蛋白表达的抑制作用呈剂量依赖性。结论: 1.外源性H2S可通过抑制VSMC对ox-LDL的内吞减少脂质在胞内的沉积。 2. H2S能够抑制VSMC内脂质沉积，其机制与其下调CD36、LOX-1表达有关。
[Abstract]:Aim: to investigate the effect of exogenous hydrogen sulfide on endocytosis low density lipoprotein (LDL) in vascular smooth muscle cells (VSMC) and its molecular mechanism in order to provide a new theoretical basis for the prevention and treatment of atherosclerosis.Methods: vascular smooth muscle cells (VSMCs) were isolated from rat abdominal aorta by tissue patch method.The culture medium was DMEM containing 10% fetal bovine serum (FBS).After 3 days of culture, the cells crawled out of the cells for 7 to 9 days for the first passage.Three or four generations of VSMC were used in the experiment.The density of 1 脳 105/ml cells was 1 ml / L ~ 3.The cells for Western blotting were inoculated in the aseptic 6-well plate, and the density of 1 脳 106/ml cells was 2 ml per pore.Experimental groups:4Western blot was used to detect the expression of CD36 blot in VSMC: it was divided into two groups: control group (80 渭 g / ml) of ox-LDL (80 渭 g / ml) NaHS2550100 渭 mol / L) group.Results:The results of 1.MTT showed that when the concentration of nahs was below 200 渭 mol/L, there was no significant effect on the activity of VSMC. When the concentration of NaHS increased to 500 渭 mol/L and 1000 渭 mol/L, the cell viability decreased obviously.2.The effects of exogenous H 2 S (using NaHS as donor) on lipid uptake of VSMC were observed by oil red O staining.The results showed that compared with the control group, the intracellular lipids increased significantly after ox-LDL was incubated alone, and the lipid deposition was more obvious when treated with CSE- 纬 -lyase inhibitor PPG, while the intracellular lipid of VSMC treated with H2S was significantly decreased.3.The endocytosis of VSMC on ox-LDL was observed with DiI-oxLDL treatment, and the accumulation of DiI-oxLDL in VSMC cells was observed.Results it was observed that H 2S could inhibit the endocytosis of ox-LDL by VSMC, while PPG enhanced the endocytosis, which was consistent with the results of oil red O staining.4.The expression of CD36 LOX-1 receptor in VSMC was detected by immunocytochemistry, and the molecular mechanism of H2S inhibiting endocytosis of ox-LDL in VSMC was preliminarily discussed.The results showed that ox-LDL could significantly induce the expression of LOX-1 receptor in VSMC. H2S could down-regulate the expression of these two receptors and up-regulate the expression of LOX-1 receptor after PPG treatment.5.In order to further determine whether the mechanism of inhibiting VSMC endocytosis by H2S is related to its down-regulation of CD36nLOX-1, the effect of NaHS on the expression of VSMC CD36 LOX-1 protein was analyzed by Western blot method.The results showed that ox-LDL significantly enhanced the expression of CD36 / LOX-1 protein induced by VSMC, while H2S inhibited the expression of two receptor proteins in a dose-dependent manner.Conclusion:1.Exogenous H 2S can reduce lipid deposition in ox-LDL by inhibiting the endocytosis of ox-LDL by VSMC.2.H2S can inhibit lipid deposition in VSMC, and its mechanism is related to its down-regulation of CD36, LOX-1 expression.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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