【作者】 吴洁；

【导师】 喻红；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 动脉粥样硬化(Atherosclerosis,As)的形成与体内低密度脂蛋白(Low-densitylipoprotein,LDL)水平有关,LDL经氧化修饰后致动脉粥样硬化的作用增强。体外实验证实As斑块中存在的三种细胞:血管内皮细胞、平滑肌细胞和巨噬细胞均可氧化低密度脂蛋白,可能是由一些氧化酶产生的氧化剂所介导,其中髓过氧化物酶发挥了至关重要作用。为了研究在细胞氧化LDL过程中髓过氧化物酶如何被激活并发挥作用,我们利用基因芯片技术在分子水平上对单核细胞和血管内皮细胞氧化LDL的可能机制进行了初步探讨。本文主要包括以下几个方面的内容: 1.将LDL与人单核细胞系THP-1和内皮细胞ECV304分别温育不同时间,检测LDL的氧化情况,研究不同时间对细胞氧化LDL的影响。 实验结果显示:氧化LDL包括MDA或共扼二烯生成随作用时间延长而增多,而且不同细胞氧化形式存在差异。从表1和表2可见THP-1单核细胞氧化LDL在9小时后降低,而内皮细胞则延迟至20小时。分别将两种细胞实验组与对照组氧化LDL生成量对时间作图,由图1和图2我们可看出单核细胞和内皮细胞氧化LDL高峰分别在9小时和20小时。由此说明两种细胞氧化LDL能力不同,单核细胞较强;作用时间对细胞氧化LDL有影响。 2.将LDL与单核细胞THP-1和内皮细胞分别温育9小时和20小时,基因芯片分析实验组与对照组信号传导相关蛋白基因表达谱的改变。 实验结果显示:经LDL诱导后,单核细胞和内皮细胞基因表达发生改变。表3列出了单核细胞中发生差异表达的基因,在1,651个检测基因中,13个基因的表达变化达到2倍以上,其中9个基因表达增加,4个表达降低。表4显示内皮细胞中差异表达的基因达到52个,23个表达增加,29个表达降低。由基因芯片扫描图(图3,5)和样品对散点图(图4,6)我们亦能发现细胞基因表达的变化。不仅如此,单核细胞和内皮细胞差异表达的的基因也有所不同,说明两种细胞可能以不同的机制氧化LDL。更多还原

【Abstract】 Atherosclerosis has been reported to be related with the level of plasma low-density lipoproteins (LDL). Once LDL was oxidized, its atherogenic effects developed. Researches demonstrated that vascular cells including macrosphages, endothelial cells and smooth muscle cells were able to oxidize low-density lipoprotein in vitro. LDL oxidation might be mediated by various oxidants generated by myeloperoxidase. To study the signaling pathway involved in cell-mediated oxidation, we observed the molecular mechanisms of LDL oxidation by using gene chip technology. This study include the following two aspects:1. LDL was cocultured with human THP-1 monocytes and endothelial cells separately for different times and LDL oxidation levels were examined.The results showed that LDL oxidation increased with the time of coculture by detecting the formation of diene or MDA and oxidation style of cells were different. As can be seen from Table 1 and 2, LDL oxidation level mediated by human THP-1 monocytes reached peak value at nine hours, but that mediated by endothelial cells delayed to twenty hours. For the sake of understanding LDL oxidation in two types of cells, we constructed to incubation time with the dispatch between Exp and control group about LDL oxidation level in these cells. From Figure 1 and 2, we could know that level of cell-mediated LDL oxidation elevated with the incubation time and reached a peak at nine hours, and twenty hours respectively. Oxidation capacity between these two types of cells was different and THP-1 had stronger ability. The incubation time also could affect LDL oxidation.2. Human THP-1 monocytes and endothelial cells were treated with LDL for nine hours and for twenty hours invidually. Microarray analysis on human signal transduction associated genes was profiled to determine changes in gene expression of two types of cells induced by LDL.From figures scanned for hybridization (Fig3,5) and scatter plot of normalized intensity data from microarray experiments (Fig4,6), we could see that many genes were differently expressed between Exp and control group. As for THP-1 monocytes, in 1,650 genes detected, thirteen genes were identified to be >two-fold difference in expression, among which nine genes with elevated expression and four genes with reduced expression. However, for endothelial cells, there were fifty-two genes different in expression: twenty-three genes expression elevated and twenty-nine genes expression reduced. We speculated that LDL oxidation mechanisms between these two types of cells were different.Conclusion: Oxidation capacity between these two types of cells was different and macrophages had stronger ability. The incubation time also could affect LDL oxidation. The mechanisms of LDL oxidation mediated by these two types of cells were different.更多还原