【作者】 蒋娜；

【导师】 朱鹏立；

【作者基本信息】 福建医科大学 ， 老年医学， 2008， 硕士

【摘要】 目的:高脂喂养建立新西兰白兔动脉粥样硬化模型。观察PPARγ、MCP-1、MMP-9及TIMP-1在兔动脉粥样硬化模型中的表达以及白藜芦醇(resveratrol)的干预作用;探讨白藜芦醇抗动脉粥样硬化(AS)可能的分子机制。方法:将70只雄性新西兰大白兔随机分为5组,分别为A组(10只):空白对照组、B组(18只):病理对照组、C组(14只):病理+Res低剂量组(4mg/Kg.d)、D组(14只):病理+Res中剂量组(8mg/Kg.d)、E组(14只):病理+Res高剂量组(16mg/Kg.d)[A组:普通饲料喂养,B、C、D、E组:高脂饲料喂养]。持续喂养12周,于0周、12周末分别抽血测定血脂,并保存血清。12周末处死所有动物,取完整胸主动脉,观察各组主动脉病理形态学变化,ELISA法测定血清中单核细胞趋化蛋白-1(MCP-1)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶抑制因子-1(TIMP-1)的蛋白表达水平,逆转录聚合酶链反应(RT-PCR)检测主动脉过氧化物酶体增殖物激活受体-γ(PPARγ)、MCP-1、MMP-9、TIMP-1的基因转录水平。结果:(1)高脂喂养12周后,病理对照组(B)血清TC、TG、LDL-C、HDL-C水平较空白对照组(A)显著增高(P<0.01),Res干预组(C、D、E)血清TG、TC、LDL-C水平明显降低(P<0.01),并呈剂量依赖性,但Res低剂量降低TC作用不明显(P>0.05);Res可升高血清HDL-C水平(P<0.01),病理+Res高剂量组较病理+Res低剂量组升高幅度更大(P<0.05)。(2)高脂喂养12周成功建立动脉粥样硬化模型。模型组主动脉弓及胸主动脉覆盖大量动脉粥样硬化斑块, Res干预组(C、D、E)可明显减轻病变程度及范围,斑块面积显著减小(P<0.01),并呈剂量依赖性。(3)病理HE染色显示,病理对照组胸主动脉上段有典型AS斑块形成,且斑块内有大量巨噬细胞浸润;Res干预组(C、D、E组)可减轻AS病变,明显抑制巨噬细胞在斑块内的浸润,病理+Res高剂量组作用更明显;空白对照组无斑块形成,血管壁未见巨噬细胞。(4)空白对照组PPARγmRNA少量表达,病理对照组表达显著升高(P<0.01);Res干预组(C、D、E)PPARγmRNA表达水平较病理对照组增高(P<0.05),并呈剂量依赖性。(5)空白对照组MCP-1、MMP-9蛋白和mRNA少量表达,病理对照组表达显著增加(P<0.01),Res干预组(C、D、E)MCP-1、MMP-9蛋白和mRNA的表达水平较病理对照组显著降低(P<0.01),并呈剂量依赖性,但仍较正常对照组显著升高(P<0.01)。(6)空白对照组TIMP-1蛋白和mRNA少量表达;病理对照组表达显著增加(P<0.01);Res干预组(C、D、E)TIMP-1蛋白和mRNA表达水平较病理对照组增高(P<0.05),呈一定的剂量依赖性。(7)AS斑块PPARγmRNA表达与MCP-1mRNA、MMP-9mRNA表达呈显著负相关(r=-0.727,r=-0.859,P均<0.01)。结论: (1)炎症反应在AS发生发展中起重要作用,包括血管壁炎症细胞浸润、循环及AS斑块内炎症介质的合成。(2)白藜芦醇具有抗AS炎症的作用,这一作用可通过降低AS兔的血清TC、TG及LDL-C水平,升高HDL-C和TIMP-1水平,抑制斑块内巨噬细胞浸润及MCP-1和MMP-9 mRNA和蛋白表达来实现,并具有一定的量效关系。(3)白藜芦醇干预AS兔12周,可上调斑块内PPARγ的基因转录水平。这为国内首次报道。更多还原

【Abstract】 Objectives: To establish a model of atherosclerosis rabbit fed with high fat diet. To investigate the expression of peroxisome proliferators-activated receptor gamma (PPARγ) in a rabbit model of atherosclerosis, and the effect of resveratrol. To explore the possible anti-atherogenic mechanism of resveratrol.Methods: seventy male New Zealand white rabbits were randomly divided into five groups: A、normal control group treated with normal sodium,B、pathological control group treated with normal sodium,C、pathological group treated with low dosage Res(4mg/ kg.d), D、pathological group treated with mid dosage Res(8mg/ kg.d), E、pathological group treated with high dosage Res(16mg/ kg.d). Rabbits in group A were fed with normal diet, Rabbits in groups B、C、D、E were fed with high fat diet. All rabbits were fed according to experiment design for 12 weeks. Serum TG, TC, LDC-C, HDL-C in rabbits were detected on the 0 and 12th week by biochemical method. The aortas were harvested for histopathological examination, and the intima-media thickness were measured. PPARγ、MCP-1、MMP-9 and TIMP-1 were examined by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbentassay (ELISA).Results: (1) The blood lipid (TC, TG, LDL-C and HDL-C ) of pathological control group is higher than normal control group (P<0.01). Administration of Res could greatly decrease the levels of serum TC, TG and LDL-C (P<0.01) in dose-dependent way, but TC was not significantly decreased in group C(P>0.05). Res could greatly increased HDL-C in dose-dependent way in group C, D and E.(2) The atherosclerosis model with rabbits had been successfully established with 12 weeks’high fat diet. The endomembranes of aortic arch and thoracic aorta in pathological control group(B) are overlaid by large area AS plaques, however there are no plaques in normal control group; Administration of Res could diminish the area of plaque(P<0.05) in dose-dependent way.(3) In pathological control group(B), there are typical AS plaques in the superior segment of thoracic aorta and lots of infiltrating macrophages in AS plaques, Administration of Res could significantly lessen AS pathological changes(P<0.05) in dose-dependent way; there are no infiltrating macrophages in normal control group.(4) The mRNA expression of PPARγin AS plaques were progressively increased in group B when comparing to group A (P<0.01). The expression of PPARγin group C, D and group E were higher than in group B (dosage dependent).(5)Compared with group A, the mRNA and protein expression of MCP-1 and MMP-9 increased significantly in group B (P<0.01); they were significantly reduced in group C, D and group E (dose dependent) (P<0.01), but still higher than in group A (P<0.01).(6)The mRNA and protein expression of TIMP-1 significantly increased in group B when compared to group A (P<0.01). Administration of Res can increased the expression of TIMP-1 which were higher than in group B (P<0.05) (dose dependent).(7)The expression of PPARγmRNA in AS plaques was negatively correlated with the expression of MCP-1 and MMP-9﹙r=-0.727, r=-0.859; p<0.01).Conclusions: (1) Inflammatory reaction is important in the development of AS, including infiltration of inflammatory cells in vessel wall, synthesis of inflammatory mediators in circulation and AS plaques.(2) Res posseses anti-inflammatory effect on AS, because it could depress the serum TC, LDL-C and TG of AS Rabbits, heighten the level of HDL-C and TIMP-1, inhibit the infiltration of macrophages and the expression of MCP-1 and MMP-9 mRNA and protein.(3) Res could up-regulate the expression of PPARγmRNA in plaques after given AS rabbits for 12 weeks.更多还原