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

发布时间：2018-06-25 10:50

本文选题：介导 + Paraoxonase　；参考：《中国协和医科大学》2007年博士论文

【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。
[Abstract]:Objective: To study the effect of Paraoxoanse (PON) gene cluster on atherosclerosis in mice and its mechanism.
Background: atherosclerosis (AS) and its complications are serious harm to human health. Oxidative low density lipoprotein (OxLDL) is an important AS factor. It not only promotes the occurrence and development of AS, but also promotes the rupture of.AS plaque in the AS plaque is an important cause of AS complications and fatal disability..OxLDL has the effect of AS on the entire arterial wall, and can also promote the whole arterial wall. Macrophages become a component of plaque and an inflammatory center.PON in the form of a gene cluster in the genome. Single PON1, PON2, and PON3 transgenes inhibit AS. by inhibiting the role of OxLDL, but the role of the whole PON gene cluster on the stability of AS, especially plaque, is not clear.
Materials and methods: a transgenic C57BL/6J mouse with a human PON gene cluster was established to hybridize to the Apo E homozygous background and feed the AS in a high fat diet for 10 or 16 weeks. After the induction, the body weight, blood fat, blood pressure and other systemic factors of the transgenic and wild control groups were first detected and then two groups of mice were killed. The intact artery was stained with oil red 0 and the degree of AS in the two groups of mice was compared with the Image Pro software. At the same time, the heart of the two groups of mice was marked with paraffin section with the aortic valve, and the HE staining was used for histological analysis. The smooth muscle cells and macrophages in the aortic root plaques were marked by immunohistochemistry, and the immune group was used in the immunological group. The expression of MMP-9 in the plaque was detected in situ, and the histological differences in the stability of the two groups were compared and analyzed. The serum of two groups of mice were detected by Elisa to detect ICAM-1 and MCP- I. At the same time, the primary macrophages of two groups of mice were examined for oxidative stress, inflammation, and foam cell formation.
Results: 5 transgenic mice with different copy numbers were established in this study. There was no obvious abnormality in the tissue expression profiles of the members of the three gene family and the endogenous PON gene cluster in mice. The body weight, blood lipid and blood sugar of the transgenic mice were not abnormal. Second transgenic mice with high copies were selected for the atherosclerosis induction test. After 10 weeks of induction, the formation of AS in female and male transgenic groups was reduced by 23.5% and 33.9%, respectively, and there was no significant difference in body weight, blood lipid, blood glucose and other systemic factors. After 16 weeks of induction, there was no significant difference in body weight, blood lipid, blood pressure and other systemic factors. The degree of AS in the female and male transgenic groups was less than that of the control group. The histological analysis showed that the proliferation of smooth muscle and the degree of macrophage infiltration in the control group were significantly more severe than that of the transgenic mice, and the abundance of MMP-9 was significantly higher than that of the transgenic mice. The fibrous cap of the transgenic plaques was thicker, the fat nuclei were smaller and the inflammatory cells were less infiltrated by the HE staining. Characteristics, the high fat induced AS plaques were more stable in the transgenic group. At the same time, it was found that the transgenic group HDL could inhibit the oxidation of LDL more significantly than the control group. Moreover, after the high fat induction of AS, the plasma MCP- I and ICAM-1 levels of the transgenic mice were significantly lower than those of the control group. The two groups of mouse peritoneal primary macrophages were compared to the OxLDL stimulation. It was found that the expression of PON gene cluster could inhibit oxidative stress, inflammation, foam cell formation and MMP-9 expression induced by stimulation.
Conclusion: the PON gene cluster can not only inhibit the serum oxLDL and its inflammatory response, but also inhibit the OxLDL induced macrophage promoting AS response, thus inhibiting the development of atherosclerosis and stabilizing the plaque. Therefore, it is possible to improve the expression and activity of the PON gene cluster as a useful means to prevent and control atherosclerosis.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R543;R-332

【共引文献】