【作者】 宋利强；

【导师】 韩梅； 温进坤；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 目的高脂血症是动脉粥样硬化(atherosclerosis, AS)的一个独立危险因素。目前认为,AS 是一种心血管系统的炎症性疾病,炎症反应贯穿了AS 的整个病理过程。经皮腔内冠状动脉成形术(percutaneous transluminal coronary angioplasty, PTCA)已广泛应用于治疗冠状动脉粥样硬化性心脏病,但术后再狭窄的发生严重影响了其中远期疗效。血管弹性回缩、内膜增生和血管重塑是PTCA后再狭窄发生的主要机制,炎症反应在内膜增生和血管重塑中起着十分重要的作用。高脂血症通过NF-κB 的激活及其介导的iNOS 的表达可促进AS 的炎症反应,但高脂血症对内皮剥脱后血管炎症反应的影响尚不清楚。因此,本实验通过建立内皮剥脱模型观察高脂血症对血管NF-κB、iNOS 蛋白表达及血管内膜增生的影响,试图证实高脂血症在血管内膜增生过程中的作用。方法1 动物模型的建立及分组将雄性SD 大鼠随机分为三组:正常组,对照组,高脂组。正常组普通饲料饮食;对照组行大鼠主动脉内皮剥脱术,普通饲料饮食;高脂组行大鼠主动脉内皮剥脱术前,用高脂饲料以15 g/只/天喂养一周,使其适应高脂饲养环境,术后继续以高脂饲料喂养。正常组大鼠于喂养后21 天、对照组及高脂组大鼠于术后21 天,从更多还原

【Abstract】 Objective Hyperlipidemia is an important risk factor for atherosclerosis (AS). AS is an inflammatory disease of cardiovascular system, and inflammation is present in the whole pathological process of AS. Percutaneous transluminal coronary angioplasty (PTCA) has been widely used for the treatment of atherosclerotic coronary artery lesions, but restenosis remains a significant limitation to the long-term efficacy of the procedure. Elastic recoil of the arterial wall, neointimal hyperplasia and vascular remodeling are the main mechanisms for post-PTCA restenosis, and inflammation plays a very important role in neointimal hyperplasia and vascular remodeling. NF-κB activation and NF-κB-mediated iNOS expression exist in the inflammation of AS caused by hyperlipidemia, but the effect of hyperlipidemia on the vascular inflammation after de-endothelialization remains unclear. To confirm the role of hyperlipidemia in the couse of neointimal hyperplasia, the present study investigated the effects of hyperlipidemia on the expression of NF-κB and iNOS as well as neointimal hyperplasia by balloon de-endothelialization. Methods 1 Establishment of de-endothelialization model and grou-ping Male SD rats were randomly divided into three groups: normal group, control group and hyperlipid group. Normal group was fed with a general diet. Control group received arterial de-endothelialization procedure and was fed with a general diet. Hyperlipid group was fed with a high-fat diet for 7 days before de-endothelialization procedure, and for 21 days after the procedure. The rats of normal group were killed at 21 days after being fed with a general diet, and those of control group and hyperlipid group were killed at 21 days after the procedure. Blood samples were taken from all rats, and serum was separated for detection of levels of lipids. The aortas were collected for section preparation, immunohistochemistry for NF-κB and iNOS, and Western blot analysis of NF-κB at the end of experiment. 2 Serum lipid levels assay We performed as kit description with fully automatic biochemical analyzer. 3 Immunohistochemical staining of NF-κB P65 and iNOS The paraffin sections were treated with xylene and alchohol. After washing with PBS, 3% H2O2 diluted with methanol was applied for 10 min to block endogeneous peroxidase, and microwave antigen retrieve for 10 min, and then blocking solution was applied for 10 min after washing. Mouse anti-rat NF-κB P65 monoclonal antibody was used as a primary antibody for staining NF-κB P65. Rabbit anti-rat iNOS polyclonal antibody was used as a primary antibody for staining iNOS. The negative control sections were treated by PBSinstead of primary antibody. Biotinylated immunoglobulin was used as a secondary antibody, and reacted for 20 min. Streptavidine horseradish peroxidase and DAB were used to detect target proteins. 4 Western blot analysis of NF-κB P65 The extracts from areries of three group rats were separated on 8% SDS-PAGE, and then blotted onto PVDF membrane. The membrane was immunologically reacted with mouse monoclonal anti-rat NF-κB P65 antibody. Results 1 Determination of the levels of serum lipids Compared with normal group and control group, the levels of serum TC, TG and LDL-C increased significantly in hyperlipid group (P < 0.01). The levels of serum HDL-C had no difference among three groups (P > 0.05). The results suggest that the high-fat diet had led to significant hyperlipidemia. 2 The morphological changes The morphological obser-vation of the aorta section showed that widespread neointimal hyperplasia and lumen stenosis were formed on day 21 after de-endothelialization, compared with smooth intima and orderly arranged media VSMC in the normal rats. At the same time, VSMC occupied the superiority in the thickened area. These results indicated that the model of rat vascular stenosis after de-endothelialization is successfully established. Moreover, foam cells were observed in the thickening neointima in hyperlipid group, suggesting that hyperlipidemia results inpathological effect更多还原