【作者】 江璐；

【导师】 刘录山；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 研究背景近年来,大量的研究表明动脉粥样硬化是一种慢性炎症性病理过程。动脉粥样硬化病变中不仅含有大量的脂质,而且有大量的炎症细胞浸润,并以单核/巨噬细胞和淋巴细胞的集聚为特征。动脉粥样硬化发生、发展中,这些炎症细胞可分泌大量的炎症因子,参与调节细胞的增殖或凋亡、细胞外基质的合成或降解以及血管的重塑等过程。PCSK9是2003年应用生物信息学方法和DNA微阵列技术发现的一个与血液胆固醇代谢调节相关的基因,最近的研究表明,PCSK9作为一种分泌性蛋白,除了降解LDLR,调节胆固醇代谢之外,可能还具有其他的一些生物学功能。我们前期研究发现PCSK9在兔动脉粥样硬化斑块以及THP-1源性巨噬细胞中高表达,LDL和ox-LDL都可以上调PCSK9在THP-1源性巨噬细胞中的表达。靶向PCSK9的RNA干扰能有效抑制ox-LDL诱导的THP-1源性巨噬细胞的凋亡作用,这提示ox-LDL在巨噬细胞中的生物学效应可以被PCSK9所影响。ox-LDL诱导巨噬细胞中炎症因子的表达与分泌是ox-LDL的重要生物学效应之一,研究PCSK9是否也能干预炎症因子的表达与分泌具有重要的科学意义。目的观察PCSK9 siRNA对ox-LDL诱导THP-1源性巨噬细胞分泌炎症因子的影响,为阐明PCSK9在动脉粥样硬化中的作用提供实验依据。材料与方法用不同浓度的ox-LDL (0,10,20,40,80μg/ml)处理经PMA诱导贴壁的THP-1源性巨噬细胞不同时间(0h,6h,12h,24h,48h),RT-PCR、ELISA、Western Blot分别检测炎症因子IL-1α、IL-6、TNF–α以及PCSK9 mRNA和蛋白的表达。应用Lipofectamine2000转染不同浓度的PCSK9 siRNA(20nmol/l,40nmol/l,80nmol/l )进入THP-1源性巨噬细胞24h,筛选出最有效的siRNA浓度,将此浓度siRNA转染THP-1源性巨噬细胞24h后,再加入ox-LDL处理24 h,RT-PCR、ELISA、Western Blot检测炎症因子IL-1α、IL-6、TNF–α表达以及NF-κB核位移。结果随着ox-LDL浓度的不断增加THP-1源性巨噬细胞炎症因子IL-1α、IL-6及TNF–αmRNA和蛋白的表达逐渐增加,同时RT-PCR、Western Blot检测发现PCSK9 mRNA、蛋白表达均增高。PCSK9 siRNA转染THP-1细胞后,再用ox-LDL处理,RT-PCR、ELISA、Western Blot检测发现与ox-LDL单独处理组相比,IL-1α、IL-6、TNF–αmRNA和蛋白表达都降低并且NF-κB核位移减少。结论PCSK9 siRNA能够抑制ox-LDL诱导的THP-1源性巨噬细胞炎症因子IL-1α、IL-6和TNF-α表达与分泌,其机制与抑制NF-κB核位移有关,提示PCSK9可能参与了炎症反应的调节。更多还原

【Abstract】 BackgroundIn recent years, a lot of research shows atherosclerosis is a chronic inflammation disease. There are not only a lot of fat, but also a large number of infiltrated inflammatory cells in atherosclerosis lesion, which is especially characterized by the accumulation of monocytes/ macrophages cell and lymphocytes. During the occurrence and development of atherosclerosis, these inflammatory cells secrete lots of inflammatory factors, which involve in the process of cell proliferation or apoptosis, extracellular matrix synthesis and degradation, vascular regeneration and so on.PCSK9, a new gene, was discovered by the application of bio-information method and DNA array technology, which is related to the regulation of blood cholesterol metabolic. Recent research indicates that PCSK9, a secretory protein, playing a critical role in the degradation of LDLR and regulation of cholesterol metabolism, may still have some other biology function. We’ve already found that pcsk9 is highly expression in rabbit atherosclerosis plaque and THP-1 macrophages, and is up-regulated by LDL and ox-LDL in macrophages. Targeted interference of PCSK9 RNA can effective inhibit ox-LDL-induced apoptosis of THP-1 cells, which indicates that the ox-LDL biology function can be influenced by PCSK9 in macrophages. An important biology effect of ox-LDL is ox-LDL-induced expression and secretion of inflammatory factors in macrophages. Whether PCSK9 can intervene the secretion of inflammatory factors has not been reported.ObjectiveTo investigate the effect of PCSK9-siRNA on inflammatory factors expression in ox-LDL-induced THP-1 macrophage cells. Materials and MethodsHuman THP-1 cells were differentiated into macrophages by the addition of 160 nmol/L phorbol 12-myristate 13-acetate (PMA) for 24h. Macrophages were incubated with diffenrent concentrations of ox-LDL (0,10,20,40,80μg/ml) for different times(0,6,12,24,48h). RT-PCR was conducted to detect IL-1α, IL-6 and TNF-αmRNA. ELISA was used for analysis of IL-1α, IL-6 and TNF-αprotein level. RT-PCR and Western Blot were conducted to detect the expression of PCSK9.The siRNA for PCSK9 were designed and synthesized, then transfected into THP-1 derived macrophages by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. The most efficient siRNA was selected to transfected into THP-1 derived macrophages, after transfection for 24h, cells were treated with ox-LDL for 24h. RT-PCR detect the expression of IL-1α, IL-6 and TNF-αmRNA, ELISA analyse IL-1α, IL-6 and TNF-αprotein level, the nuclear translocation of NF-κB was detect by Western Blot.ResultsIn THP-1 derived macrophages, ox-LDL increased IL-1α, IL-6 and TNF-αmRNA and protein expression. PCSK9 was upregulated with increasing concentration of ox-LDL, while 80μg/ml ox-LDL increased significantly. The RNA interference experiment showed that PCSK9 siRNA was successfully transfected into cells and 80 nmol/L as effectively suppressed dose of siRNA was selected by RT-PCR and Western Blot. Compared with 80μg/ml ox-LDL treatment group,the suppression of inflammation factors and NF-κB nuclear translocation in THP-1 derived macrophages transfected with 80 nmol/L siRNA for 24h and incubated with 80μg/ml ox-LDL for another 24h was detected by RT-PCR ,ELISA and Western Blot.ConclusionsPCSK9 siRNA can inhibit ox-LDL induced inflammatory factor expression, such as IL1-α,IL-6 and TNF-αin THP-1 macrophage. Its mechanism is related to the inhibition of NF-κB nuclear translocation, which indicate that PCSK9 may be involved in the regulation of inflammatory reaction.更多还原