敲低前蛋白转化酶枯草溶菌素9(PCSK9)抑制人主动脉内皮细胞的内皮间质转化

Knockdown of PCSK9 inhibits endothelial-to-mesenchymal transition of human aortic endothelial cells

目的以人主动脉内皮细胞(HAEC)为研究对象,探讨敲减前蛋白转化酶枯草溶菌素9(PCSK9)对β甘油磷酸、地塞米松、L-抗坏血酸诱导内皮间质转化(EndoMT)的保护作用。方法以(0、10、30、50)mmol/Lβ甘油磷酸联合100 nmol/L地塞米松和50μg/ml L-抗坏血酸诱导HAEC建立EndoMT模型。采用PCSK9的特异性小干扰RNA(si-PCSK9)转染HAEC,实时荧光定量PCR与Western blot法检测HAEC的PCSK9的mRNA和蛋白表达。将HAEC随机分为空白组、EndoMT组、转染阴性对照小干扰RNA的EndoMT组、转染si-PCSK9的EndoMT组。实时荧光定量PCR检测α平滑肌肌动蛋白(α-SMA)、成纤维细胞特异蛋白1(FSP1)、血管内皮钙黏蛋白(VE-cadherin)的mRNA水平,Western blot法检测α-SMA、VE-cadherin的蛋白水平,免疫荧光染色法检测α-SMA的表达。结果30 mmol/Lβ甘油磷酸诱导EndoMT效果最佳,发生EndoMT时,PCSK9的mRNA及蛋白表达上调。而PCSK9敲减后,α-SMA、FSP1的表达下调,VE-cadherin的表达上调。结论敲低PCSK9可抑制HAEC的EndoMT。

Objective To investigate the protective effect of proprotein convertase subtilisin/kexin type 9(PCSK9)knockdown on endothelial-to-mesenchymal transition(EndoMT)induced byβglycerophosphate,dexamethasone,and L-ascorbic acid in human aortic endothelial cells(HAECs).Methods EndoMT model was established by inducing HAECs with(0,10,30,50)mmol/L ofβglycerophosphate combined with 100 nmol/L of dexamethasone and 50μg/mL of L-ascorbic acid.HAECs were transfected with specific small interfering RNA of PCSK9(si-PCSK9),and the mRNA and protein expression levels of PCSK9 in HAECs were detected by real-time quantitative PCR and Western blotting.HAECs were randomized into blank group,EndoMT group,EndoMT group transfected with negative control small interfering RNA(si-NC),and EndoMT group transfected with si-PCSK9.The mRNA levels ofα-smooth muscle actin(α-SMA),fibroblast-specific protein 1(FSP1),and vascular endothelial cadherin(VE-cadherin)were detected by real-time quantitative PCR,the protein levels ofα-SMA and VE-cadherin were detected by Western blotting,and the expression ofα-SMA was detected by immunofluorescence staining.Results 30 mmol/L ofβglycerophosphate had the best effect in inducing EndoMT,and the expression of PCSK9 mRNA and protein was up-regulated when EndoMT occurred.After PCSK9 knockdown,the expressions ofα-SMA and FSP1 were down-regulated,while the expression of VE cadherin was up-regulated.Conclusion Knockdown of PCSK9 inhibits the EndoMT of HAECs.