氧化型低密度脂蛋白诱导内皮细胞凋亡中前蛋白转化酶枯草溶菌素9与炎症因子的关系

Relationship between PCSK9and inflammation factor expression in ox-LDL-induced endothelial cells apoptosis

目的观察氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)凋亡中前蛋白转化酶枯草溶菌素9(PCSK9)与炎症因子表达的关系。方法用ox-LDL处理HUVECs 0小时、12小时、24小时、36小时、48小时,分别检测PCSK9、白细胞介素6(IL-6)、单核细胞趋化蛋白1(MCP-1)、基质金属蛋白酶9(MMP-9)、C反应蛋白(CRP)mRNA和蛋白的表达以及核因子-κB(NF-κB)核位移。Lipofectamine 2000转染PCSK9小分子干扰RNA(PCSK9siRNA)进入HUVECs 6小时后,加入ox-LDL处理24小时,分别检测PCSK9、IL-6、MCP-1、MMP-9、CRP的表达以及NF-κB核位移。结果随着ox-LDL处理时间的增加,PCSK9、IL-6、MCP-1、MMP-9、CRP mRNA的表达上调、NF-κB的核位移明显增加,以24小时表达(核位移)最明显,随后表达(核位移)逐渐减少(P<0.05);细胞培养上清液中IL-6、MCP-1、MMP-9、CRP蛋白的浓度随着时间的推移逐渐增加,48小时达到峰值,与0小时比较,48小时增加最明显(P<0.01)。siRNA组PCSK9、IL-6、MCP-1、MMP-9、CRP mRNA和蛋白的表达以及NF-κB核位移较阴性转染组明显降低(P<0.01)。结论 PCSK9siRNA能够抑制ox-LDL诱导的HUVECs炎症因子的表达,PCSK9可能参与了炎症反应的调节。

Objective To investigate the relationship between proprotein convertase subtilisin/kexin type 9（PCSK9）and inflammatory factor expression in apoptosis of human umbilical vein endothelial cells（HUVECs）induced by oxidized low density lipoprotein（ox-LDL）.Methods HUVECs were incubated with ox-LDL for different times（0h,12 h,24h,36 h,48h）.The mRNA and protein expression of PCSK9,inter leukin 6（IL-6）,monocyte chemotactic protein-1（MCP-1）,matrix metalloprotein-9（MMP-9）,C-reactive protein（CRP）and the nucler displacement of nucler factor-kappa B（NF-κB）were detected.The siRNA for PCSK9 was designed and synthesized,then was transfected into HUVECs by Lipofectamine 2000.After transfection for 6h,cells were treated with ox-LDL for 24 h,the mRNA and protein expression of PCSK9,IL-6,MCP-1,MMP-9,CRP and the nucler displacement of NF-κB were detected.Results With the increase of ox-LDL processing time,the mRNA expressions of PCSK9,IL-6,MCP-1,MMP-9and CRP were significantly higher,and the nucler displacement of NF-κB was significantly increased,especially for 24 h incubation,and the subsequent expression and the nucler displacement were gradually decreased（P〈0.05）.The protein concentration of IL-6,MCP-1,CRP and MMP-9in cell culture supernatant increased gradually over time,and reached the peak at 48 h.Compared with the 0hgroup,the 48 hgroup increased most obviously（P〈0.01）.The mRNA and protein expressions of PCSK9,IL-6,MCP-1,MMP-9,CRP and the nucler displacement of NF-κB in siRNA group were significantly lower than those in negative transfection group（P〈0.01）.Conclusion PCSK9 siRNA can inhibit inflammation factor expression in apoptosis of HUVECs induced by ox-LDL.And PCSK9 may be involved in the regulation of inflammation.