BAC介导人Paraoxonase基因簇转基因鼠系的建立及其对动脉粥样硬化的影响

【作者】 折志刚；

【导师】 梁植权； 刘德培； 韦玉生；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 目的:研究Paraoxoanse(PON)基因簇对小鼠动脉粥样硬化的作用及其机制背景:动脉粥样硬化(AS)及其并发症严重危害人类健康。氧化性低密度脂蛋白(OxLDL)是重要的致AS因子,不但促进AS的发生和发展,还促进AS斑块的破裂。AS斑块破裂是AS并发症及致死致残的重要原因。OxLDL对整个动脉壁都有致AS作用,也可促使巨噬细胞成为斑块的成分和炎症中心。PON在基因组中以基因簇的形式存在,单个PON1、PON2和PON3转基因通过抑制OxLDL的作用而抑制AS。但是整个PON基因簇对AS尤其是斑块稳定性的作用还不明确。材料和方法:研究建立了人PON基因簇的转基因C57BL/6J小鼠,杂交到Apo E纯合缺失背景,并以高脂饮食分别喂食10周或16周诱导AS的发生。诱导结束后首先检测并比较转基因组和野生对照组的体重、血脂、血压等系统因素,之后处死两组小鼠取其完整动脉进行油红0染色并以Image Pro软件进行双盲定量比较两组小鼠AS发生程度。同时取两组小鼠的心脏以主动脉瓣为标记进行石蜡切片,HE染色进行组织学分析,免疫组化标记主动脉根部斑块的平滑肌细胞和巨噬细胞,并以免疫组化原位检测斑块内MMP-9的表达,比较分析两组斑块在稳定性方面的组织学差别。取两组小鼠的血清以Elisa检测ICAM-1及MCP-Ⅰ。同时在细胞水平,取两组小鼠的腹腔原代巨噬细胞进行氧化应激、炎症、泡沫细胞形成检测。结果:本研究共建立5株不同拷贝数的转基因小鼠,转入的三个基因家族成员的组织表达谱与小鼠内源性PON一致。PON基因簇转基因鼠无明显异常,体重、血脂、血糖也无异常。选择高拷贝的第二株转基因小鼠进行动脉粥样硬化诱导实验。经过10周诱导,发现雌性和雄性转基因组AS形成程度分别比对照组减少23.5%和33.9%,而其体重、血脂、血糖等系统因素没有明显差异。经过16周诱导,在体重、血脂、血压等系统因素无明显差异的同时,雌性和雄性转基因组AS程度分别比对照组减少33.3%和27.7%,组织学分析发现对照组小鼠的斑块内不但平滑肌增殖和巨噬细胞浸润程度明显比转基因组小鼠严重,而且MMP-9的丰度显著高于转基因组,结合HE染色显示示的转基因组斑块中纤维帽较厚、脂核较小及炎性细胞浸润较少的特点,提示的高脂诱导的AS斑块在转基因组中更具稳定性。同时发现转基因组HDL较对照组能更显著地抑制LDL的氧化。而且高脂诱导AS后,转基因组小鼠的血浆MCP-Ⅰ和ICAM-1水平明显低于对照组。比较两组小鼠腹腔原代巨噬细胞对OxLDL刺激的反应,发现PON基因簇的表达可以抑制刺激导致的氧化应激、炎症、泡沫细胞形成及MMP-9的表达。结论:PON基因簇不但可以抑制血清oxLDL及其导致的炎症反应,而且可以抑制OxLDL诱导的巨噬细胞促AS反应。从而抑制动脉粥样硬化的发生发展,并稳定斑块。因此,提高PON基因簇的表达和活性可能成为防治动脉粥样硬化的有用手段。更多还原

【Abstract】 Targets-Elucidtion functions and corresponding mechanisms of Paraoxoanse (PON) gene cluster on atherosclerosis(AS) in mice.Background- Oxidized LDL(OxLDL) plays very important roles in AS,which not only initiates and ptomotes AS,but also sitmulates the AS plaque to go to rupture.Rupture of AS plaque is the main cause of death resulted from AS. OxLDL induces AS via acting on the whole aorta,it can also promote the macrophages to become components of plaque and center of inflammation.PON exists as a gene cluster in the genome and its member PON1,PON2 and PON3 had been testified being able to inhibit atherogenesis by acting on oxLDL.But effects of the whole cluster on AS,expecially on plaque stability,still remain to be elucidated.Methods - PON cluster transgenic(PC Tg) mice on C57BL/6J background were generated,crossed to apoE KO background,and induced AS with high fat diet for 10 weeks or 16 weeks.After induction,body weight,serum lipid and sugar indexes and blood pressure were detected and compared the transgenic and wild-type mice.Then we sacrificed these mice,took out the intact aortas and detected the lesion area of the whole aortas with Oil Red O staining.The lesion areas were quilified with Image Pro software and compared the two groups.At the same time,hearts of the two proups were collected and sectioned at the level of aortic root for further histologic analysis.We analyzed these sections morphologically using HE staining,marked smooth muscle cells and macrophages using immunohistochemistry,detected expression of MMP-9 in situ in the sections using immunohistochemistry,and compared these data between two groups in the aspect of plaque stability.Levels of serum ICAM-1 and MCP-Ⅰwere detected using Elisa.ROS generation assay,inflammation detection and foam cell formation experiments were carried out to explore corresponding mechanisms in the MPMs from transgenic mice and wilt-type mice.Results- Five independent PC Tg mouse lines on C57BL/6J background were generated.Expression pattern of transgenic human PONs was the same with intrinsic mouse PONs.No obvious abnormality characters including body weight, serum lipid and sugar indexes were boserved in the transgenic mice.P2 strain transgenic mice were selected to establish AS models.After 10 weeks of induction,lesion area of transgenic mice aorta decreased 23.5%and 33.9 %compared to control in female and male groups,respectively,while the body weight and high level of serum cholesterol indexes were similar in the two groups.After 16 weeks of induction,lesion area of transgenic mice aorta decreased 33.3%and 27.7%%compared to control in female and male groups, respectively,with the similar body weight and high level of serum cholesterol indexes.Histological analysis revealed that formed plaque in PC TG mice exhibited thicker fibro cap,smaller necrotic core and less infiltration of inflammation cell,thus suggesting that formed plaque intransgenic mice were more stable prone.These results were further convinced by phenomenon of less proliferation of vascular smooth muscle cells,less infiltration of macrophages and lower levels MMP-9 expression in PC TG mice.PC transgene enhanced the ability of HDL to protect LDL against oxidation and inhibited serum level of MCP-Ⅰand ICAM-1.Pro-atherogenic reaction of mice peritoneal macrophages(MPM) induced by oxLDL were significantly inhibited by PC TG as indicated by less ROS generation,inflammation,MMP-9 expression and foam cell formation.Conclusion- Human PON cluster transgene repressed and stabilized atherosclerosis plaque in vivo via acting not only on serum oxidized LDL but also on vascular macrophage cell.PONs may become useful targets for AS treatment.更多还原