【作者】 程恒辉；

【导师】 阮秋蓉；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 背景:脱氢表雄酮(Dehydroegiandrosterone,DHEA)是性激素合成过程的中间产物,是血浆中浓度最高的类固醇激素。近年来,DHEA引起了医学界的广泛注意,尤其是其抗衰老作用越来越引起人们的重视。大量的流行病学及动物实验均显示,DHEA具有较好的预防和治疗动脉粥样硬化(Atherosclerosis,AS)作用。己有研究报道,DHEA在细胞内由细胞色素P450芳香酶降解为雌酮后发挥作用。因此有很多学者认为DHEA的抗AS作用是通过转化为雌激素或者雄激素而发挥的。但是也有部分学者研究发现,DHEA可能通过其自身的生物学活性发挥抗AS作用。这一颇具争议性的问题目前尚无定论。目的:本实验通过建立兔动脉粥样硬化模型,研究DHEA对高脂饲养的兔动脉粥样硬化发展过程中的作用以及维甲酸受体激动剂—全反式维甲酸对这一过程有无促进作用。同时,原代培养大鼠血管平滑肌细胞,通过脂质体瞬时转染细胞色素P450芳香酶基因(CYP19)的方法进一步探讨DHEA抗动脉粥样硬化的作用机制是否与细胞色素P450芳香酶催化DHEA转化为雌雄激素的作用有关。方法:将新西兰雄性大耳白兔进行分笼分组喂养,给予高脂饮食构建兔动脉粥样硬化模型,通过对兔主动脉形态学观察,血脂水平测量,斑块部位血管内皮细胞血管细胞粘附分子(VCAM-1)及单核细胞趋化蛋白-1(MCP-1)表达的逆转录聚合酶链反应(RT-PCR)及免疫细胞化学检测来研究DHEA的抗动脉粥样硬化作用。原代培养SD雄性大鼠血管平滑肌细胞(VSMCs),当细胞纯度达95%以上,取第3～4代细胞用于实验。采用脂质体瞬时转染的方法将CYP19转染至VSMCs中, 24小时后加入氧化低密度脂蛋白(Oxidized Low Density Lipoprotein, ox-LDL)及DHEA,并通过RT-PCR、Realtime PCR及ELISA的方法观察并检测不同组别VCAM-1和MCP-1的表达情况。结果:(1)DHEA可降低雄性成年兔低密度脂蛋白胆固醇(LDL-C)水平,但对血清总胆固醇(TC)、血清甘油三酯(TG)和高密度脂蛋白胆固醇(HDL-C)无明显影响。(2)DHEA干预后兔的主动脉粥样硬化斑块面积与总面积的比值、内膜厚度、内膜中膜比显著低于未用药的高脂饲养组。(3)免疫组织化学及RT-PCR结果均显示DHEA能显著降低兔VCAM-1、MCP-1的表达。(4)动物实验未发现全反式维甲酸明显的促进DHEA抑制高脂饲养的新西兰大耳白兔AS的作用。(5)转染CYP19后的VSMCs经ox-LDL刺激后其表达VCAM-1和MCP-1的水平与ox-LDL刺激未转染CYP19的VSMCs无明显区别。结论:DHEA能够抑制兔动脉粥样斑块的形成,降低VCAM-1和MCP-1的分泌。而这一抗动脉粥样硬化的作用并不通过其转化为雌激素或者雄激素而发挥,很有可能与其自身的生物学活性有关。更多还原

【Abstract】 Background Dehydroepiandrosterone (DHEA) is the middle outcome in sex hormone synthesis and is the most abundant androgen produced primarily by the adrenal gland. Resent years, much attention is paid on DHEA, especially its antiatherosclerotic effect. Huge of epidemic investigation and animal experiments all demonstrated that it has excellent preventive and therapeutic effect on atherosclerosis(AS). There have been reported that DHEA exerts its antiatherosclerotic effect by transformation into estrogen. So many scholars thought the antiatherosclerotic effect of DHEA is just by transformation. But other scholars found out that the antiatherosclerotic effect of DHEA is performed by the bioactivity of its own. There has no verdict on this disputable problem.Objective To observe the function of DHEA on rabbit atherogenisis and the relationship with all-trans retinoic acid, a retinoic acid acceptor activator, in this progress. Meanwhile, culture primary vascular smooth muscle cells from rats, by the method of tanscient transfected cytochrome P450 aromatase to demonstrate whether the antiatherosclerotic effect of DHEA is related to its conversion to estrogen or androgen and investigate whether its mechanism has something to do with the catalysis of the cytochrome P450 aromatase (CYP19).Methods Male New Zealand White (NZW) rabbits were divided into 5 groups and individually caged. And the rabbits were fed with high cholesterol diet to make model. The serum lipid levels were measured. The aortas were sampled for morphological observation. The mRNA levels of vascular cell adhesion molecule-1 (VCAM-1)、monocyte chemoattractant protein-1(MCP-1) were detected by RT-PCR technic and immunocytochemistry. Rat aortic SMCs were prepared from 4-6 week old SD rat. When the purity of cells achieves 95%, the cells of the third or the fourth passage were used in experimentation. The expression of MCP-1 mRNA was determined by RT-PCR. The plasmid with CYP19 was transient transfected into cultured VSMCs. 24 hours later, the cells were stimulated with Oxidized Low Density Lipoprotein(ox-LDL) and DHEA, the gene and protein of VCAM-1 and MCP-1 expression level of each group were detected using the method of RT-PCR, Realtime PCR, ELISA.Results (1) The level of LDL- cholesterol(LDL-C) in male rabbits’serum could be reduced by DHEA, but total cholesterol (TC), triglyceride (TG) and HDL- cholesterol(HDL-C) have no obvious infection. (2) The ratio of atherosclerotic plaque area to the total area of aortas in the group fed with HCD(high cholesterol diet) and DHEA was obvious decreased compared with the group just fed with HCD(p<0.05). (3) The results of immunohistochemistry and RT-PCR all revealed that DHEA can obviously reduce the expression of VCAM-1 and MCP-1. (4) In the animal experiment, after all-trans retinoic acid was added, the change of each index wasn’t obvious (p>0.05). (5) There wasn’t obvious difference of VCAM-1 and MCP-1 expression in the groups of transfected plasmid with or without CYP19 (p>0.05).Conclusions DHEA could retard atherosclerotic plaque formation in high cholesterol-fed rabbits and downregulate the expression of VCAM-1 and MCP-1. This antiatherosclerotic effect of DHEA doesn’t work by transforming into estrogen or androgen which may be clearly with its own bioactivity.更多还原