干扰小RNA沉默α1抗胰蛋白酶对类风湿关节炎成纤维细胞的作用探讨  

Analysis of the effect of siRNA silencing α1 antitrypsin on rheumatoid arthritis fibroblasts

作　　者：赵燕[1,2] 曹盛楠[3] 孙国栋 潘继红[1] 常晓天[4] 孟庆松[5] Zhao Yan;Cao Shengnan;Sun Guodong;Pan Jihong;Chang Xiaotian;Meng Qingsong(Shandong Medicinal Biotechnology Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, China;Shandong Normal University, College of Life Sciences, Jinan 250014, China;Department of Rehabilitation Medicine, Affiliated Hospital of Shandong Academy of Medical Sciences, Jinan 250031, China;Laboratory Department, Shandong Provincial Qianfoshan Hospital, the First Hospital Affiliated with Shandong First Medical University, Jinan 250014, China;Medical Laboratory Center, Shandong Provincial Qianfoshan Hospital, the First Hospital Affiliated with Shandong First Medical University, Jinan 250014, China)

机构地区：[1]山东省医药生物技术研究中心基础实验室,山东第一医科大学(山东省医学科学院),济南250062 [2]山东师范大学生命科学学院,250014 [3]山东省医学科学院附属医院康复医学科,250031 [4]山东第一医科大学第一附属医院(山东省千佛山医院)检验科,济南250014 [5]5山东第一医科大学第一附属医院(山东省千佛山医院)医学实验中心,济南250014

出　　处：《中华风湿病学杂志》2019年第9期617-622,I0003,共7页Chinese Journal of Rheumatology

基　　金：山东省自然科学基金(ZR2014YL040).

摘　　要：目的探讨干扰小RNA(siRNA)沉默α1抗胰蛋白酶(α1-AT)基因对类风湿关节炎成纤维样滑膜(RA-SFs)生物学行为的影响。方法取5例RA患者的膝关节滑膜组织原代培养,采用人工合成的沉默α1-AT siRNA,特异性抑制α1-AT在RA-SFs中的表达;瞬时转染24、36 h后,采用RT-qPCR法检测抑制效率,同时检测α1-AT基因沉默后各相关基因的表达;采用四甲基偶氮唑蓝(MTT)法、Transwell小室、细胞划痕、ELISA等方法检测干扰α1-AT表达后对细胞增殖、侵袭、迁移的影响和IL-17,TNF-α,IL-1α,IL-1β等相关炎性因子分泌的表达;观察通路抑制剂[ERK抑制剂、信号转导和转录激活因子3(STAT3)抑制剂、NF-κB抑制剂]刺激细胞时,对α1-AT的影响变化。多组间比较采用单因素方差分析,LSD-t检验及χ^2检验。结果与阴性对照组相比,siRNA α1-AT组转染24 h时,α1-AT mRNA水平抑制效果显著(P<0.05),且RA SFs的增殖速率未受影响(P>0.05),细胞的侵袭和迁移能力显著下降(P<0.05);IL-17、IL-1β分泌略微降低(P>0.05),TNF-α和IL-1α分泌略微增加(P>0.05);α1-AT下游基因MMP-2-169、Hu-Bax1、MMP-9、Hu Bax和Hu Bcl-xl表达均明显下降(P<0.05)。信号通路抑制剂ERK抑制剂(PD98059)和NF-κB抑制剂(PDTC)对α1-AT均有显著性影响(P<0.05)。结论α1-AT基因沉默对RA-SFs的生物学行为具有一定潜在作用,进一步研究该机制有利于为RA的治疗提供证据支持。Objective To investigate the effect of siRNA silencing α1 antitrypsin (α1-AT) gene on the biological behavior of rheumatoid arthritis fibroblast-like synoviocytes (RA-SFs). Methods Primary culture of knee synovial tissue from 5 patients with rheumatoid arthritis (RA) was performed. The artificially synthesized silencing α1-AT siRNA specifically inhibits the expression of α1-AT in RA-SFs. After 24 and 36 hours of transient transfection, the inhibition efficiency was detected by Quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the expression of related genes after α1-AT gene silencing was detected.Furthermore, ethyl thiazolyl tetrazolium (MTT) assay, Trans-well chamber, cell scratch and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of interfering α1-AT expression on cell proliferation, invasion and migration, and secretion of interleukin (IL)-17, Tumor necrosis factor (TNF)-α, IL-1α, IL-1β and other related inflammatory factors. At the same time, when the pathway inhibitor (ERK inhibitor, signal transducer and activator of transcription 3 (STAT3) inhibitor, NF-κB inhibitor) stimulated cells, the effect on α1-AT was changed. One-way analysis of variance was used for comparison between the two groups;further pairwise comparison using LSD-t test;The count data was checked by χ^2 test. Results Compared with the negative control group, the siRNA α1-AT group was transfected for 24 hours, the α1-AT mRNA level was significantly inhibited (P<0.05), and the proliferation rate of RA-SFs was not affected (P>0.05), and the invasion and migration ability of cells decreased significantly (P<0.05). The secretion of IL-17 and IL-1β was slightly decreased (P>0.05), while TNF-α and IL-1α were not affected (P>0.05). In addition, the expression of α1-AT downstream genes Matrix metallop roteinases (MMP)-2 169, Hu-Bax1, MMP-9, Hu Bax and Hu Bcl-xl were significantly decreased (P<0.05). Moreover, the signaling pathway inhibitors ERK inhibitor (PD98059) and NF-κB inh

关 键 词：Α1抗胰蛋白酶 关节炎 类风湿 成纤维细胞 RNA 小分子干扰 

分 类 号：R593.22[医药卫生—内科学]