本文選題：介導 + Paraoxonase　； 參考：《中國協(xié)和醫(yī)科大學》2007年博士論文

【摘要】： 目的:研究Paraoxoanse(PON)基因簇對小鼠動脈粥樣硬化的作用及其機制 背景:動脈粥樣硬化(AS)及其并發(fā)癥嚴重危害人類健康。氧化性低密度脂蛋白(OxLDL)是重要的致AS因子,不但促進AS的發(fā)生和發(fā)展,還促進AS斑塊的破裂。AS斑塊破裂是AS并發(fā)癥及致死致殘的重要原因。OxLDL對整個動脈壁都有致AS作用,也可促使巨噬細胞成為斑塊的成分和炎癥中心。PON在基因組中以基因簇的形式存在,單個PON1、PON2和PON3轉(zhuǎn)基因通過抑制OxLDL的作用而抑制AS。但是整個PON基因簇對AS尤其是斑塊穩(wěn)定性的作用還不明確。 材料和方法:研究建立了人PON基因簇的轉(zhuǎn)基因C57BL/6J小鼠,雜交到Apo E純合缺失背景,并以高脂飲食分別喂食10周或16周誘導AS的發(fā)生。誘導結(jié)束后首先檢測并比較轉(zhuǎn)基因組和野生對照組的體重、血脂、血壓等系統(tǒng)因素,之后處死兩組小鼠取其完整動脈進行油紅0染色并以Image Pro軟件進行雙盲定量比較兩組小鼠AS發(fā)生程度。同時取兩組小鼠的心臟以主動脈瓣為標記進行石蠟切片,HE染色進行組織學分析,免疫組化標記主動脈根部斑塊的平滑肌細胞和巨噬細胞,并以免疫組化原位檢測斑塊內(nèi)MMP-9的表達,比較分析兩組斑塊在穩(wěn)定性方面的組織學差別。取兩組小鼠的血清以Elisa檢測ICAM-1及MCP-Ⅰ。同時在細胞水平,取兩組小鼠的腹腔原代巨噬細胞進行氧化應激、炎癥、泡沫細胞形成檢測。 結(jié)果:本研究共建立5株不同拷貝數(shù)的轉(zhuǎn)基因小鼠,轉(zhuǎn)入的三個基因家族成員的組織表達譜與小鼠內(nèi)源性PON一致。PON基因簇轉(zhuǎn)基因鼠無明顯異常,體重、血脂、血糖也無異常。選擇高拷貝的第二株轉(zhuǎn)基因小鼠進行動脈粥樣硬化誘導實驗。經(jīng)過10周誘導,發(fā)現(xiàn)雌性和雄性轉(zhuǎn)基因組AS形成程度分別比對照組減少23.5%和33.9%,而其體重、血脂、血糖等系統(tǒng)因素沒有明顯差異。經(jīng)過16周誘導,在體重、血脂、血壓等系統(tǒng)因素無明顯差異的同時,雌性和雄性轉(zhuǎn)基因組AS程度分別比對照組減少33.3%和27.7%,組織學分析發(fā)現(xiàn)對照組小鼠的斑塊內(nèi)不但平滑肌增殖和巨噬細胞浸潤程度明顯比轉(zhuǎn)基因組小鼠嚴重,而且MMP-9的豐度顯著高于轉(zhuǎn)基因組,結(jié)合HE染色顯示示的轉(zhuǎn)基因組斑塊中纖維帽較厚、脂核較小及炎性細胞浸潤較少的特點,提示的高脂誘導的AS斑塊在轉(zhuǎn)基因組中更具穩(wěn)定性。同時發(fā)現(xiàn)轉(zhuǎn)基因組HDL較對照組能更顯著地抑制LDL的氧化。而且高脂誘導AS后,轉(zhuǎn)基因組小鼠的血漿MCP-Ⅰ和ICAM-1水平明顯低于對照組。比較兩組小鼠腹腔原代巨噬細胞對OxLDL刺激的反應,發(fā)現(xiàn)PON基因簇的表達可以抑制刺激導致的氧化應激、炎癥、泡沫細胞形成及MMP-9的表達。 結(jié)論:PON基因簇不但可以抑制血清oxLDL及其導致的炎癥反應,而且可以抑制OxLDL誘導的巨噬細胞促AS反應。從而抑制動脈粥樣硬化的發(fā)生發(fā)展,并穩(wěn)定斑塊。因此,提高PON基因簇的表達和活性可能成為防治動脈粥樣硬化的有用手段。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R543;R-332

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