银屑病患者血浆中前蛋白转化酶枯草溶菌素9的表达及对外周血CD4^+T细胞活化的影响

Expression of proprotein convertase subtilisin/kexin type 9 in the plasma of patients with psoriasis and its effect on the activation of peripheral CD4^+T cells

目的通过检测银屑病患者血浆中前蛋白转化酶枯草溶菌素9(PCSK9)的含量及其对外周血CD4^+T细胞分泌干扰素γ(IFN-γ)、白细胞介素17A(IL-17A)的影响,探讨PCSK9在银屑病发病机制中的作用。方法招募2016年2月至2017年12月于中国医学科学院皮肤病医院门诊就诊的30例寻常性银屑病患者和30例健康志愿者(对照组)。患者组中男16例,女14例,年龄18~66岁,病程1个月至15年。抽取受试者外周静脉血,分离血浆和单个核细胞(PBMC),实时荧光定量PCR(q-PCR)检测PBMC中PCSK9mRNA表达,酶联免疫吸附实验(ELISA)检测血浆中PCSK9蛋白浓度。磁珠法分离每例受试者外周血CD4^+T细胞,并均分为两组,实验组加入PCSK9蛋白共培养,对照组不加PCSK9蛋白,培养24h后,ELISA检测培养基上清液中IFN-γ和IL-17A的表达水平。两组间比较采用独立样本t检验,银屑病患者血浆PCSK9浓度与银屑病皮损面积和严重程度指数(PASI)的关系采用Pearson相关性分析。结果银屑病组和对照组PBMC中未检测到PCSK9mRNA的表达。银屑病组血浆中PCSK9表达[(243.58±11.91)μg/L]显著高于对照组[(199.74±31.09)μg/L],差异有统计学意义(t=5.761,P<0.001)。银屑病患者外周血CD4^+T细胞与PCSK9蛋白共培养后,IFN-γ和IL-17A表达水平[分别为(6150.00±212.13)ng/L和(1532.00±11.31)ng/L]均高于未加PCSK9蛋白的对照组[分别为(4650.00±212.13)ng/L和(698.5±266.58)ng/L],差异均有统计学意义(t=7.071和4.418,均P<0.05)。健康对照CD4^+T细胞实验组和对照组均未检测到IFN-γ和IL-17A表达。结论银屑病患者血浆中PCSK9含量增高,可能通过活化外周血CD4^+T细胞参与银屑病的发病机制。

ObjectiveTo investigate the role of proprotein convertase subtilisin/kexin type 9(PCSK9)in the pathogenesis of psoriasis by detecting the level of PCSK9 in the plasma of patients with psoriasis and evaluating its effect on the secretion of interferon gamma(IFN-γ)and interleukin-17A(IL-17A)by peripheral CD4^+T cells.MethodsTotally,30 outpatients with psoriasis vulgaris and 30 healthy volunteers(controls)were enrolled from Hospital for Skin Diseases,Chinese Academy of Medical Sciences between February 2016 and December 2017.Of the 30 patients,16 were males,and 14 were females.Their age varied from 18 to 66 years,and the course of disease ranged from 1 month to 15 years.Peripheral venous blood samples were obtained from the patients and controls,and the plasma and peripheral blood mononuclear cells(PBMC)were isolated.Real-time fluorescence-based PCR(q-PCR)was performed to measure mRNA expression of PCSK9 in the PBMC,and enzyme-linked immunosorbent assay(ELISA)to determine the concentration of PCSK9 in the plasma.Peripheral CD4^+T cells were isolated from the PBMC by magnetic bead method,and divided into 2 groups to be co-cultured with(experiment group)or without PCSK9 protein(control group).After 24-hour treatment,ELISA was conducted to detect the levels of IFN-γand IL-17A in the culture supernatant.Statistical analysis was carried out by using two-sample t test for the comparison between the two groups,and Pearson correlation analysis for analyzing correlations between the plasma level of PCSK9 and psoriasis area and severity index(PASI)score in the patients with psoriasis.ResultsPCSK9 mRNA expression was undetected in the PBMC of the patients with psoriasis and controls.The plasma level of PCSK9 was significantly higher in the patients with psoriasis([243.58±11.91]μg/L)than in the healthy controls([199.74±31.09]μg/L,t=5.761,P<0.001).After co-culture of the peripheral CD4^+T cells from patients with PCSK9 protein,the levels of IFN-γand IL-17A both significantly increased([6 150.00±212.13]ng/L,[1 532.00±11.31]ng/L,respectively)compared with the control group co-cultured without PCSK9 protein([4 650.00±212.13]ng/L,[698.5±266.58]ng/L,respectively;t=7.071,4.418 respectively,both P<0.05).IFN-γand IL-17A were undetected in the culture supernatant of CD4^+T cells from the healthy controls in the experiment group or control group.ConclusionThe plasma level of PCSK9 increases in patients with psoriasis,which may be involved in the pathogenesis of psoriasis by activating peripheral CD4^+T cells.