【作者】 武春艳；

【导师】 刘录山；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 研究背景内膜损伤是动脉粥样硬化(Atherosclerosis, As)的始发环节。研究表明oxLDL引起平滑肌细胞凋亡,可能与上调P53和下调Bcl-2的表达有关。另一些研究已证实oxLDL与细胞膜表面的受体LOX-1结合引起内皮细胞的凋亡,主要经下调Bcl-2的蛋白表达,线粒体依赖性凋亡信号通路。PCSK9作为一种神经细胞凋亡调节转化酶,参与肝脏的再生,调节神经细胞的凋亡,脂质代谢等多种作用。通过降低肝细胞上LDLR的数量,影响LDL的内化,使血液中LDL不能清除,从而导致高胆固醇血症。高胆固醇血症是一个已被确认的动脉粥样硬化的独立危险因素。另有研究证实PCSK9在胚胎发育中具有促进神经细胞分化的作用,体外细胞实验发现其可参与神经细胞的凋亡,在此过程中有凋亡调节因子Caspase3和死亡受体6的共同调节。本课题组的前期研究表明:PCSK9 siRNA能明显抑制oxLDL诱导的THP-1源性巨噬细胞的凋亡,但其机制不清。PCSK9 siRNA是否对血管内皮细胞的凋亡也有影响还未见报道。目的本研究为了检测oxLDL诱导的HUVECs凋亡中PCSK9的表达变化;PCSK9 siRNA是否抑制oxLDL诱导的HUVECs凋亡及探讨其机制。材料与方法用不同浓度的oxLDL处理HUVECs 24h,Hoechst33258染色检测细胞凋亡,RT-PCR、western blot分别检测PCSK9 mRNA、LOX-1 mRNA和NARC-1、LOX-1蛋白的表达。应用Lipofectamine2000转染不同浓度的PCSK9 siRNA进入HUVECs,筛选出最有效的siRNA浓度转染HUVECs 24 h后,再加入oxLDL处理24 h,Hoechst33258染色观察评价细胞凋亡,western blot检测凋亡相关蛋白Bcl-2,Bax及Caspase3,-8,-9的表达,酶标法检测Caspase3和Caspase9的活性,Hoechst33258染色观察形态评价细胞凋亡和流式细胞计数凋亡率。结果随着oxLDL浓度的不断增加,HUVECs凋亡率逐渐增加,以80μg/ml oxLDL处理24h后,Hoechst33258染色可见大量凋亡细胞。同时RT-PCR、western blot检测发现LOX-1和NARC-1mRNA,蛋白表达均增高。不同浓度的PCSK9 siRNA转染HUVECs后,RT-PCR和western blot筛选出siRNA终浓度,作用细胞24 h后,再用oxLDL处理24 h,Hoechst33258染色和流式细胞术检测oxLDL处理组细胞凋亡率明显增加,转染PCSK9 siRNA组细胞凋亡率明显减少。而RT-PCR和western blot检测结果表明LOX-1的蛋白和mRNA表达在转染PCSK9 siRNA组和oxLDL组无明显差异。Western blot检测结果发现PCSK9 siRNA能明显下调Bax蛋白的表达和上调Bcl-2蛋白的表达,同时观察转染80nmol/L PCSK9 siRNA后用oxLDL处理细胞24小时其对Caspase3,Caspase8和Caspase9的影响,western blot结果显示Caspase3,Caspase9的蛋白表达明显上调,而对Caspase8的蛋白表达无影响。酶标法检测Caspase3,Caspase9的活性均降低。结论1.OxLDL能上调PCSK9 mRNA和蛋白质的表达,而PCSK9 siRNA能有效抑制PCSK9基因的表达,从而抑制由oxLDL诱导的HUVECs凋亡。2.PCSK9 siRNA抑制HUVECs的凋亡主要经Bcl/Bax-Cyt C-Caspase 9-Caspase3线粒体凋亡通路。更多还原

【Abstract】 BackgroundEndothelial injury is an initiating factor of atherosclerogenesis. Endothelial cells apoptosis is easier than necrosis leading to the occurrence of atherosclerosis. Studies have shown that smooth muscle cell apoptosis induced by oxLDL may be related to upregulate the expression of P53 and downregulate the expression of Bcl-2. Other studies have confirmed that oxLDL can bind LOX-1 receptor, reduce the expression of Bcl-2 and can inhibit apoptosis induced by a mitochondrial-dependent apoptotic signaling pathway.PCSK9 as a neural apoptosis-regulated convertase 1, plays an important role in liver regeneration, the differentiation of cortical neurons and lipid metabolism, and other multiple roles by impacts the level of low density lipoprotein receptor (LDLR) in liver.However, excessive accumulation of intracellular LDL affects the clearance of plasma LDL,then resulting in hypercholesterolemia.It has been confirmed that hypercholesterolemia is an independent atherosclerotic risk factor. Scientists have discovered that PCSK9 facilitated the neuronal differentiation in the process of embryonic development, and found to participate in the apoptosis of nerve cells in vitro, apoptotic regulatory factors Caspase3 and co-regulated death receptor 6 may be regulate this process. Our research have showed that PCSK9 siRNA inhibited oxLDL induced THP-1 derived-macrophage apoptosis, but the mechanism is unclear. Whether PCSK9 siRNA also impact apoptosis of endothelial cells have not been reported.ObjectiveIn order to examine the effect of oxLDL on PCSK9/NARC-1 expression in HUVECs, and examine the role of PCSK9 siRNA on oxLDL induced apoptosis of HUVECs and the expression of apoptotic-related proteins. Materials and MethodsHUVECs were incubated with diffenrent concentration of oxLDL for 24h.The apoptosis of HUVECs was observed by staining with Hoechst33258; RT-PCR、western blot were conducted to detect the expression of PCSK9、LOX-1 mRNAs and proteins respectively. The PCSK9 siRNAs gene were transfected into HUVECs by positive ion liposome Lipofectamine 2000. Transfection efficiency was assessed by fluorescence microscope assay. The most efficient siRNA was selected to transfected into HUVECs,after transfection for 24 h, cells were treated with oxLDL for 24 h, the rate of HUVECs apoptosis transfected siRNA was detected by staining with Hoechst 33258 and flow cytometer. Western blot was conducted to detect the expression of apototic proteins Bcl-2,Bax,Caspase3,8,9 expressions. The activity of Caspase3,9 was detected by Enzyme linked immunosorbent assay.ResultsThe results showed that a number of cells with nuclear condensation induced by 80μg/ml oxLDL for 24h increased significantly,Hoechst33258 staining showed a number of cells apoptosis. PCSK9 was upregulated with increasing concentration of oxLDL in HUVECs, while 80μg/ml oxLDL increased significantly. Moreover,RT-PCR、western blot showed that LOX-1,PCSK9 mRNAs and proteins both increased.The different concentration of PCSK9 siRNAs were transfected into HUVECs,the most effective dose of siRNA was selected by RT-PCR and western blot.HUVECs pretreated with PCSK9 siRNA for 24h , then cells treated with oxLDL for 24h, oxLDL-induced apoptosis was significantly reduced in HUVECs transfected with siRNA detected by staining with Hoechst33258 and flow cytometer. PCSK9 siRNA upregulated Bcl-2 protein expression while Bax expression was decreased. Compared with control,PCSK9 siRNA supressed Caspase3,9 proteins expression,but had on effect on Caspase8. Moreover,activity of Caspase 3,9 both decreased by PCSK9 siRNA.Apopotosis of HUVECs decreased significantly by PCSK9 siRNA,but the expression of LOX-1 between siRNA and oxLDL treatment had no difference. Conclusions1.These results revealed that the expression of PCSK9 mRNA and protein were increased by oxLDL. PCSK9 gene expression could be effectively suppressed by siRNA. So oxLDL induced apoptosis of HUVECs could be effectively suppressed by PCSK9 siRNA.2.Our study demostrated that PCSK9 siRNA inhibits HUVECs apoptosis via Bcl/Bax-Cyt C-Caspase 9-Caspase3 mitochondrial pathway.更多还原