摘要
目的:探讨内质网到细胞核信号1(endoplasmic reticulum-to-nucleus signaling 1,ERN1)基因对炎症微环境中软骨细胞炎症因子和软骨分解代谢的影响。方法:通过CRISPR-Cas9系统构建人ERN1基因敲除的C28/I2软骨细胞株(ERN1 KO);从C57BL/6J背景的ERN1软骨特异性敲除(ERN1 cKO)小鼠软骨组织分离原代软骨细胞,分别为对照组(ERN1flox/flox)、ERN1 cKO组(ERN1flox/flox-Col2Cre+);在C28/I2人正常软骨细胞中过表达ERN1腺病毒(Ad ERN1),以AdGFP为对照组;实验采用10μg/L白介素1β(interleukin-1β,IL-1β)诱导过表达ERN1软骨细胞或ERN1缺陷软骨细胞,形成炎症微环境,用qPCR和Western blot检测IL-1β处理ERN1缺失或过表达ERN1细胞后,肿瘤坏死因子α(tumor necrosis factor α,TNFα)、IL-4、IL-6、IL-10等炎症因子和软骨分解代谢标志物基质金属蛋白酶13(matrix metalloproteinase 13,MMP13)、含血小板结合蛋白基序的解整联蛋白及金属蛋白酶5(a disintegrin and me?talloproteinase with thrombospondin motifs 5,ADAMTS5)的表达。前交叉韧带切除(anterior cruciate ligament resec?tion,ACLT)术制作ERN1 cKO小鼠骨关节炎(osteoarthriti,OA)模型,免疫组化法检测软骨组织TNFα和IL-1β的表达。结果:成功将ERN1单向导RNA(sgRNA)构建至LentiCRISPRv2载体,并在C28/I2细胞中成功筛选出ERN1敲除稳定细胞株。qPCR结果显示,在体外炎症微环境中,敲除ERN1可上调软骨细胞促炎细胞因子IL-6和TNFαmRNA水平(P<0.05),下调抗炎细胞因子IL-4和IL-10 mRNA水平(P<0.05)。Western blot结果显示,当软骨细胞处于炎症微环境中,敲除ERN1可显著上调TNFα表达(P<0.05),增强ADAMTS5和MMP13的表达(P<0.05),而过表达ERN1则显著抑制TNFα表达(P<0.05)。免疫组化法结果显示,ACLT术后ERN1 cKO可促进TNFα、IL-1β表达。结论:ERN1基因通过调节促炎及抗炎因子平衡参与炎症反应。
AIM:To explore the effect of endoplasmic reticulum-to-nucleus signaling 1 (ERN1) gene on the biological properties of chondrocytes in an inflammatory microenvironment.METHODS:The ERN1 knockout C28/I2 hu-man normal chondrocyte cell line ERN1 KO was constructed by CRISPR-Cas9 system.Primary chondrocytes from ERN1cartilage-specific knockout mice with C57BL/6J background were isolated,and the experiments were divided into control group (ERN1flox/flox),ERN1 cKO group (ERN1flox/flox-Col2Cre+).ERN1 adenovirus (Ad ERN1) was over-expressed in C28/I2human normal chondrocytes,with AdGFP as the control group.Interleukin-1β (IL-1β) at 10μg/L was used in chondro-cytes to form an inflammatory microenvironment.The expression of inflammatory factors tumor necrosis factor α (TNFα),IL-4,IL-6 and IL-10,and cartilage catabolism markers matrix metalloproteinase 13 (MMP13) and a disintegrin and me-talloproteinase with thrombospondin motifs 5 (ADAMTS5) in chondrocytes after ERN1 deletion or overexpression in the in-flammatory microenvironment induced by IL-1β was detected by qPCR and Western blot.RESULTS:The ERN1 sgRNA was successfully constructed into LentiCRISPRv2 vector,and ERN1 knockout stable cell line were successfully screened in C28/I2 cells.qPCR results showed that in the inflammatory microenvironment in vitro,ERN1 deficiency up-regulated the mRNA levels of pro-inflammatory cytokines IL-6 and TNFα in chondrocytes (P<0.05),and down-regulated the mRNA levels of anti-inflammatory cytokines IL-4 and IL-10 (P<0.05).Western blot results showed that when chondro-cytes were in the inflammatory microenvironment,ERN1 deficiency significantly up-regulated the expression of pro-inflam-matory cytokine TNFα (P<0.05),and enhanced the expression of cartilage catabolism markers ADAMTS5 and MMP13(P<0.05).The expression of TNFα was significantly inhibited after over-expression of ERN1 (P<0.05).CONCLU-SION:ERN1 regulates the inflammatory sensitivity of chondrocytes by regulating the levels of pro-inflammatory and antiinflammatory factors,
作者
梁利
邓琳
罗瑞
冯乃波
李小丽
范梦恬
郭风劲
LIANG Li;DENG Lin;LUO Rui;FENG Naibo;LI Xiaoli;FAN Mengtian;GUO Fengjin(Department of Cell Biology and Genetics,Chongqing Medical University,Chongqing 400016,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第2期314-324,共11页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81871769)
重庆市科委面上项目(No.cstc2020jcyj-msxmX0175)
重庆市科委博士后科学基金项目(No.cstc2021jcyj-bshX0214)
重庆医科大学研究生拔尖人才培育项目(No.BJRC202019)。
