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背景:研究表明,轻度修饰低密度脂蛋白与动脉粥样硬化的形成有关,除了具有蓄积低密度脂蛋白作用外还有很强的生物学活性,可在培养细胞及动物体内诱导巨噬细胞集落刺激因子等多种生物活性物质的表达。目的:采用差异显示-聚合酶链反应技术研究轻度修饰低密度脂蛋白诱导血管内皮细胞的基因表达差异,为进一步阐明轻度修饰低密度脂蛋白与动脉粥样硬化的关系奠定基础。设计:重复测量设计。单位:泰山医学院。材料:实验于2003-07/2004-07在泰山医学院基础研究所完成。人脐静脉内皮细胞培养基为M199,在37℃,50mL/L的CO2条件下培养。细胞生长到融合状态时,培养基中加入轻度修饰低密度脂蛋白,至终浓度为400mg/L,诱导处理30h。方法:用差异显示反转录-聚合酶链反应技术分析轻度修饰低密度脂蛋白诱导下人血管内皮细胞的基因表达差异,并用反向Northern分析证实差异显示基因片段。主要观察指标:①内皮细胞mRNA的差异显示分析。②差异片段的克隆、序列分析及同源性比较。③诱导与非诱导小鼠肝脏mRNA的反向Northern分析。结果:轻度修饰低密度脂蛋白诱导下人血管内皮细胞出现一些上调和下调的基因片段。上调基因有胸腺素β4、FGFRI原癌基因伴随蛋白、FK506结合蛋白、rTSβ蛋白和细胞间粘附分子1;下调的基因有Apobec-1结合蛋白1、细胞色素B561和ERP72。结论:用差异显示反转录-聚合酶链反应方法证实在体外轻度修饰低密度脂蛋白可诱导人脐静脉内皮细胞一些基因的表达水平变化,引起轻度修饰低密度脂蛋白血管内皮细胞发生病理变化,最终导致动脉粥样硬化斑块的形成。
Background: Studies have shown that mildly modified LDL is associated with the formation of atherosclerosis. In addition to having the effect of accumulating LDL, it has strong biological activity and can induce macrophages in cultured cells and animals Colony stimulating factor and other bioactive substances expression. OBJECTIVE: To study the difference of gene expression induced by mildly modified low density lipoprotein in vascular endothelial cells by differential display-polymerase chain reaction (PCR), and to lay a foundation for elucidating the relationship between mildly modified low density lipoprotein and atherosclerosis. Design: repeat measurement design. Unit: Taishan Medical College. Materials: The experiment was performed at the Institute of Basic Medical Sciences, Taishan Medical College from July 2003 to July 2004. Human umbilical vein endothelial cell culture medium M199, at 37 ℃, 50mL / L of CO2 culture conditions. When the cells grew to the confluent state, mild modified low density lipoprotein was added to the medium to a final concentration of 400 mg / L for 30 hours. Methods: Differential display RT - PCR was used to analyze the gene expression differences of human vascular endothelial cells induced by lightly modified low density lipoprotein. The reverse transcription - northern analysis was used to confirm the differential display of gene fragments. MAIN OUTCOME MEASURES: ① The difference of endothelial cell mRNA was analyzed. ② cloning of different fragments, sequence analysis and homology comparison. ③ Reverse Northern analysis of induced and non-induced mouse liver mRNA. RESULTS: Some mildly-modified low-density lipoprotein induced some up-and down-regulated gene fragments in human vascular endothelial cells. Up-regulated genes were thymosin β4, FGFRI protooncogene concomitant protein, FK506 binding protein, rTSβ protein and intercellular adhesion molecule-1; down-regulated genes were Apobec-1 binding protein 1, cytochrome B561 and ERP72. Conclusion: Differential display reverse transcription - polymerase chain reaction method was used to confirm the mild modification of low density lipoprotein in vitro can induce the expression of some genes in human umbilical vein endothelial cells, resulting in mildly modified low density lipoprotein vascular endothelial cell Pathological changes eventually lead to the formation of atherosclerotic plaques.