【作者】 张红明；

【导师】 何作云；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 研究背景动脉粥样硬化（atherosclerosis,AS）发病机制的研究已经经历了一个多世纪,主要围绕4种学说:脂肪浸润学说、血小板聚集和血栓形成学说、平滑肌细胞克隆学说和损伤反应学说,但至今详细发病机理仍未完全阐明。近年来不少研究发现,动脉粥样硬化的病理表现具有炎症病理的基本表现形式:变质、渗出和增生,其形成过程中也会出现类似类风湿性关节炎、慢性胰腺炎和肝硬化等慢性炎症性疾病的细胞间相互作用,随着炎症细胞和炎症介质的不断检出,动脉粥样硬化不再被认为是单纯的动脉壁脂质堆积的疾病,而是进展性炎症反应,符合炎症表现的普遍规律。目前,在动脉粥样硬化发现的促炎因子较多,如血小板源性生长因子β（PDGF-β）、血小板衍生因子（PDEF）、单核细胞趋化蛋白1、白细胞介素3、环氧合酶-2、基质金属蛋白酶、去组合蛋白、脂肪酸结合蛋白等,由于目前临床针对上述介质的抗动脉粥样硬化药物起效缓慢且疗效有待提高,因此是否还有其它炎症因子参与动脉粥样硬化进展有待进一步研究。FIZZ1（Found in Inflammatory Zone 1）是2000年新发现的一个与炎症相关的缺氧诱导有丝分裂因子,是运用表达序列标记数据库扫描的方法发现的一种分泌型蛋白,分子量9.4KD,具有刺激肺动脉平滑肌细胞增殖、收缩血管、促进血管新生、刺激肌纤维母细胞分化等功能,在脂肪组织的间质血管以及选择性激活的巨噬细胞中均有表达,缺氧的肺血管壁也有表达,在实验性肺纤维化、哮喘、过敏性肺炎、寄生虫感染等疾病中具有重要意义,其在动脉粥样硬化中是否表达则为未知,由于动脉粥样硬化斑块内存在Th2型细胞因子以及大量激活的巨噬细胞,动脉粥样硬化的血管壁也存在缺氧,因此,我们推测在动脉粥样硬化斑块内可能有FIZZ1表达。本实验用免疫组织化学方法检测动脉粥样硬化斑块内FIZZ1表达,RT-PCR检测FIZZ1mRNA表达,并探讨FIZZ1对体外培养的平滑肌细胞增殖,平滑肌细胞清道夫受体SR-A,巨噬细胞CD36表达影响,同时探讨降脂治疗对动脉粥样硬化斑块FIZZ1表达的影响。研究方法本研究分三部分,第一部分:选取C57BL/6J ApoE基因敲除鼠及C57BL/6J野生型小鼠,分别喂食高脂饲料及普通饲料,24周后处死小鼠,石蜡包埋血管后作连续切片,行HE染色及FIZZ1免疫组化染色,检测斑块FIZZ1表达情况, RT-PCR检测动脉粥样硬化斑块内FIZZ1mRNA表达,然后,模拟粥样斑块内细胞因子环境,用Th2型细胞因子刺激培养的平滑肌细胞以及巨噬细胞,RT-PCR以及激光共聚焦显微镜观察刺激后细胞FIZZ1mRNA及其蛋白表达情况,初步鉴定粥样斑块内分泌FIZZ1的可能的细胞;第二部分用终浓度分别为3×10^-6mmol/L,9×10^-6mmol/L,2.7×10^-5mmol/L的FIZZ1刺激培养的平滑肌细胞,3H掺入法以及MTT法测定FIZZ1对平滑肌细胞增殖的影响;用ox-LDL以及终浓度分别为3×10^-6mmol/L , 9×10^-6mmol/L , 2.7×10^-5mmol/L的FIZZ1刺激培养的平滑肌细胞,蛋白质印迹技术确认SR-A表达后,激光共聚焦显微镜对SR-A表达进行定位,用流式细胞术检测FIZZ1对平滑肌细胞SR-A及其巨噬细胞CD36表达的影响,评价FIZZ1能否影响动脉粥样硬化吞噬脂质的关键受体。第三部分:制作动脉粥样硬化以及高脂血症动物模型,然后用辛伐他汀干预实验动物,21周后,用免疫组化方法检测粥样斑块FIZZ1蛋白表达情况,RT-PCR方法检测FIZZ1mRNA表达情况,同时检测动脉粥样硬化斑块形态及其血脂水平,探讨调脂治疗对FIZZ1表达的影响。结果1.ApoE基因敲除鼠高脂饲养24周后,主动脉根部明显形成动脉粥样硬化,斑块体积大,免疫组化可见FIZZ1在动脉粥样硬化斑块内明显表达,RT-PCR可见FIZZ1mRNA表达,同龄野生型C57BL/6J鼠正常血管壁内,未见FIZZ1表达,Th2型细胞因子能刺激培养的巨噬细胞表达FIZZ1,但不能刺激体外培养的平滑肌细胞表达FIZZ1。2.MTT检测平滑肌细胞增殖提示,FIZZ1 3×10^-6mmol/L组与对照组吸光度值比较,明显升高,P<0.05,差异有显著性;FIZZ1 9×10^-6mmol/L组与FIZZ1 3×10^-6mmol/L组比较,吸光度值明显升高,差异有显著性,P<0.05, FIZZ1 2.7×10^-5mmol/L组与9×10^-6mmol/L组比较,吸光度值明显升高,差异有显著性,说明FIZZ1能刺激平滑肌细胞增殖。3H-TdR掺入法检测也提示FIZZ1刺激平滑肌细胞增殖。3.激光共聚焦显微镜可见在ox-LDL刺激24小时的平滑肌细胞,有SR-A表达,主要位于细胞膜,蛋白质印迹技术发现ox-LDL能促进平滑肌细胞SR-A表达。4.流式细胞术检测提示,FIZZ1 3×10^-6mmol/L组与对照组SR-A表达阳性率比较,统计学上有极显著性差异（P<0.01）;FIZZ1 9×10^-6mmol/L组与FIZZ1 3×10^-6mmol/L组SR-A表达阳性率比较,有显著性差异（P<0.05）, FIZZ1 2.7×10^-5mmol/L组与9×10^-6mmol/L组SR-A表达阳性率比较,有极显著性差异（P<0.01）,说明FIZZ1能明显促进ox-LDL刺激的平滑肌细胞SR-A表达。5.ox-LDL明显刺激巨噬细胞CD36表达,FIZZ1不影响体外培养的巨噬细胞清道夫受体CD36的表达,说明FIZZ1不能通过影响巨噬细胞CD36表达来影响巨噬细胞ox-LDL的摄取,影响动脉粥样硬化。6.ApoE基因敲除鼠高脂饲养21周后,主动脉根部明显形成动脉粥样硬化,血脂代谢明显紊乱,免疫组化可见FIZZ1在粥样硬化斑块内明显表达,辛伐他汀能降低动脉粥样硬化斑块内FIZZ1mRNA及其蛋白表达（与对照组比较,差别具有统计学意义,P<0.05）。结论1.FIZZ1在ApoE基因敲除鼠粥样斑块内明显表达,同龄野生型C57BL/6J鼠血管壁内,FIZZ1不表达,FIZZ1可能为一种新的影响动脉粥样硬化进展的促炎因子。2.Th2型细胞因子可以刺激巨噬细胞FIZZ1表达,但不能刺激平滑肌细胞FIZZ1表达,Th2型细胞因子刺激巨噬细胞可能为斑块内FIZZ1表达机理之一。3.FIZZ1明显刺激体外培养的主动脉平滑肌细胞增殖。4.FIZZ1不影响体外培养的巨噬细胞清道夫受体CD36的表达,FIZZ1能促进ox-LDL诱导的体外培养的平滑肌细胞SR-A表达,从而可能加速平滑肌细胞吞噬脂质,促进动脉粥样硬化进展。5.辛伐他汀能明显减少动脉粥样硬化斑块内FIZZ1表达,减轻动脉粥样硬化,此可能为调脂药物抗动脉粥样硬化的非调脂作用之一。更多还原

【Abstract】 Background and ObjectivesThe study of pathogenesis of atherosclerosis （AS） has lasted more than one century. There are four hypotheses: lipid infiltration hypothesis, platelet aggregation （PA） and thrombosis hypothesis, vascular smooth muscle cell （VSMCs） clone hypothesis and response-to-injury hypothesis. In recent years, AS has been identified to resemble some fundamental pathological manifestations of inflammation including alteration, exudation and proliferation. In the development of AS, cell-cell interaction is similar to that in chronic inflammation diseases such as rheumatoid arthritis, chronic pancreatitis, hepaticcirhosis, etc. With the identifying of inflammatory cells and mediators, AS is no longer considered as a simple disease of fatty substance accumulation in the artery wall, but as a progressive inflammatory reaction, which is consistent with the general rule of inflammation. Based on his response-to-injury hypothesis, professor Ross proposed that AS is an inflammation disease. Presently, many inflammatory mediators in AS have been discovered such as PDGF-β,bFGF, TGF-βand VEGF.FIZZ1 （Found in Inflammatory Zone 1） was discovered by EST in 2000 and identified to be a new hypoxia derivation mitgenic factor associated with inflammation. It is a secreting protein, with the molecular weight of 9.4KD, which has the function of enhancing the proliferation of pulmonary artery VSMCs, contracting blood vessel, promoting the neogenesis of blood vessel, and stimulating myofibroblast differentiation. FIZZ1 can be detected in the stromal blood vessel of adipose tissue and alternatively activated macrophages, as well as anoxic pulmonary vessel wall. Because there are Th2 cytokines and generous alternatively activated macrophages in atherosclerotic plaque, and atherosclerotic vessel wall is usually hypoxic, we presume that FIZZ1 may exist in atherosclerotic plaque. In this study, we detected the expression of FIZZ1 in atherosclerotic plague by immunohistochemistry method, explored the effect of FIZZ1 on the proliferation of VSMCs and the expression of VSMCs scavenger receptor A （SR-A）, and investigated the effect of Simvastatin on the expression of FIZZ1 in atherosclerotic plaque. MethodsThe first part: sixteen C57BL/6J ApoE-/- mice were fed with high fat diet while 12 C57BL/6J mice were fed with normal diet. Twenty-four week later, all the mice were executed for the collection of aortas which were dissected from aortic root to abdominal aorta. Then the aortic sections were imbedded with paraffin for HE dying and FIZZ1 immunohistochemistry. At the same time, the expression of FIZZ1 mRNA in atherosclerotic plaque was detected by RT-PCR. In vitro, after Th2 cytokine was used to stimulate cultural VSMCs and macrophage, the expression of FIZZ1 mRNA and protein were detected by RT-PCR and laser confocal microscopy, respectively. The second part: The cultural VSMCs were stimulated with FIZZ1 at different final concentrations of 3×10^-6mmol/L, 9×10^-6mmol/L and 2.7×10^-5mmol/L respectively. Then the effect of FIZZ1 on the proliferation of VSMCs was detected by ³H-TdR incorporation and MTT. After the cultural VSMCs were stimulated with ox-LDL and FIZZ1 at different final concentrations of 3×10^-6mmol/L, 9×10^-6mmol/L, 2.7×10^-5mmol/L, respectively. Laser confocal microscopy was applied to ascertain and locate the expression of SR-A. Western-blot and flow cytometry were used to detect the effect of FIZZ1 on the expression of SR-A in VSMCs, and meanwhile, flow cytometry was used to detect the effect of FIZZ1 on the expression of CD36 in macrophages.The third part: The animal models of AS and hyperlipoidemia were established, and then interfered with Simvastatin. 21 weeks later, the effect of Simvastatin on the expression of FIZZ1 was evaluated by RT-PCR and immunohistochemistry. The morphology of atherosclerotic plaque was observed.Results1. After 24 weeks, AS emerged obviously in the aortic root in ApoE-/- mice fed with high fat diet, and the area of plaque was fairly large. The expression of FIZZ1 was detected in atherosclerotic plaque by immunohistochemistry and RT-PCR. In the normal artery of C57BL/6J wild type mice, FIZZ1 was negative. Th2-type cytokine can induce the expression of FIZZ1 in macrophages, but not in VSMCs.2. The proliferation of VSMCs was detected by MTT. Compared to the control group, the A value of FIZZ1 3×10^-6 mmol/L group increased obviously （P<0.05）. Compared to FIZZ1 3×10^-6 mmol/L group, the A value of FIZZ1 9×10^-6 mmol/L group increased obviously （P<0.05）. Compared to FIZZ1 9×10^-6 mmol/L group, the A value of FIZZ1 2.7×10^-5 mmol/L group increased obviously （P<0.05）. These results indicated FIZZ1 enhance aorta VSMCs proliferation, which was also observed by 3H-TdR incorporation.3. SR-A positive expression was found in VSMCs treated with ox-LDL after 24 hours, which located mainly in cell membrane by laser confocal microscopy. SR-A protein was also detected by Western - blot.4. There were significant differences in SR-A positive rates between FIZZ1 3×10^-6 mmol/L group and the control group by flow cytometry （P<0.01）. The same results were found between FIZZ1 9×10^-6 mmol/L group and FIZZ1 3×10^-6 mmol/L group, between FIZZ1 2.7×10^-5 mmol/L group and FIZZ1 9×10^-6 mmol/L group, too. These results indicate FIZZ1 enhance the expression of SR-A induced by ox-LDL in VSMCs.5. FIZZ1 had no effect on the expression of scavenger receptor CD36 in cultured macrophages, which reveals that it couldn’t interfere with the ox-LDL uptake of macrophages and atherosclerosis.6. After 21 weeks, AS emerged obviously in the aortic root in ApoE-/- mice fed with high fat diet, and lipid metabolism in the mice disordered. FIZZ1 in atherosclerotic plaque was positive by immunohistochemistry. Simvastatin could decrease the expression of FIZZ1 mRNA and protein in atherosclerotic plaque （P<0.05）.Conclusions1. FIZZ1 can express in the atherosclerotic plaque in C57BL/6J ApoE-/- mice, but not in the normal artery of C57BL/6J wild type mice.2. Th2-type cytokine can enhance the expression of FIZZ1 in mcrophages, but not in VSMCs. Th2-type cytokine is one of the probable factors which induce the expression of FIZZ1 in atherosclerotic plaque.3. FIZZ1 can stimulate the proliferation of VSMCs obviously.4. FIZZ1 had no effect on the expression of scavenger receptor CD36 in cultured macrophages. FIZZ1 can promote the development of AS by the up-regulation of the expression of SR-A and the phagocytose of fatty substance in VSMCs.5. Simvastatin can decrease the area of plaque and the expression of FIZZ1 in atherosclerotic plaque obviously, which may be a mechanism of anti-AS drug.更多还原