沉默NLRP3基因对促炎剂诱导大鼠脑微血管内皮细胞表达炎症因子的影响研究  

Effect of NLRP3 gene silencing on expression of proinflammatory agents-induced inflammatory factors in rat brain microvascular endothelial cells

作　　者：张静 潘晓华[2] 刘国荣 郭立志[2] 倪朝文[2] 王越 于英英 ZHANG Jing;PAN Xiaohua;LIU Guorong;GUO Lizhi;NI Zhaowen;WANG Yue;YU Yingying(Baotou Clinical College,Inner Mongolia Medical University,Baotou,Inner Mongolia 014040,P.R.China;Department of Neurology,Baotou Eighth Hospital,Baotou,Inner Mongolia 014040,P.R.China;Department of Neurology,Baotou Central Hospital,Baotou,Inner Mongolia 014040,P.R.China)

机构地区：[1]内蒙古医科大学包头临床医学院,内蒙古包头014040 [2]包头市第八医院神经内科,内蒙古包头014040 [3]包头市中心医院神经内科,内蒙古包头014040

出　　处：《华西医学》2022年第6期855-862,共8页West China Medical Journal

基　　金：内蒙古自治区自然科学基金(2018MS08055);内蒙古自治区卫生健康委员会医疗卫生科技计划项目(202201529);内蒙古医科大学联合项目(YKD2021LH064)。

摘　　要：目的研究沉默NOD样受体家族含pyrin结构域蛋白3(NOD-like receptor family,pyrin domain containing protein 3,NLRP3)基因对大鼠脑微血管内皮细胞(brain microvascular endothelial cell,BMEC)经促炎剂脂多糖(lipopolysaccharide,LPS)和腺苷三磷酸(adenosine triphosphate,ATP)诱导产生炎症因子的影响,及NLRP3炎症小体信号通路是否在BMEC经促炎剂作用形成的脑小血管病细胞模型中发挥作用。方法体外提取雄性Wistar大鼠BMEC并鉴定内皮细胞的形态和纯度。将正常培养的BMEC分为空白对照组和LPS+ATP组,利用蛋白质印迹法和实时聚合酶链反应检测NLRP3炎症小体及下游炎症因子胱天蛋白酶(Caspase)-1的表达,采用独立样本t检验进行组间比较;采用小干扰RNA(small interfering RNA,siRNA)分子对BMEC内的特定基因NLRP3进行沉默,将siRNA NLRP3和siRNA质粒阴性对照分别转染BMEC后,再将转染后的细胞分为4组,即siNC组(未沉默目的基因,不加促炎剂)、siNLRP3组(沉默目的基因,不加促炎剂)、siNC+LPS+ATP组(未沉默目的基因,加促炎剂)、siNLRP3+LPS+ATP组(沉默目的基因,加促炎剂),利用蛋白质印迹法和实时聚合酶链反应检测各组NLRP3和Caspase-1的表达,采用析因设计的方差分析进行组间比较。结果大鼠BMEC分离培养24 h后脑微血管段呈“串珠状”,48 h后“岛屿状”细胞团形成,72 h后“铺路石”样单层细胞贴壁生长,后细胞逐渐密集达汇合度80%。免疫荧光染色检测BMEC阳性率达96%。在正常培养的细胞中,LPS+ATP组NLRP3和Caspase-1的蛋白及mRNA表达较空白对照组升高(P<0.05)。在RNA干扰培养的细胞中,siNLRP3组较siNC组、siNLRP3+LPS+ATP组较siNC+LPS+ATP组NLRP3、Caspase-1的蛋白及mRNA表达量均降低(P<0.05),siNC+LPS+ATP组较siNC组、siNLRP3+LPS+ATP组较siNLRP3组NLRP3、Caspase-1的蛋白及mRNA表达量均升高(P<0.05);对于NLRP3、Caspase-1的mRNA相对表达量,给予转染质粒和促炎剂干预的交互效应有统计学意义(P<0Objective To study the effect of silencing the NOD-like receptor family,pyrin domain containing protein 3(NLRP3)gene on the production of inflammatory factors induced by lipopolysaccharide(LPS)and adenosine triphosphate(ATP)in rat brain microvascular endothelial cells(BMECs),and whether NLRP3 inflammasome signaling pathway plays a role in the BMEC model of cerebral small vessel disease induced by proinflammatory agents.Methods BMECs from male Wistar rats were extracted in vitro and the morphology and purity of endothelial cells were identified.BMECs in normal culture were divided into blank control group and LPS+ATP group.The expression levels of NLRP3 inflammasome and downstream inflammatory factor Caspase-1 were detected by Western blot and real-time polymerase chain reaction,and compared by student’s t test between the two groups.Small interfering RNA(siRNA)was used to silence the specific gene NLRP3 in BMECs.After transfection of siRNA NLRP3 and siRNA plasmid negative control into BMECs,the transfected cells were divided into four groups,namely,siNC group(non silenced target gene),siNLRP3 group(silenced target gene),siNC+LPS+ATP group(non silenced target gene and added proinflammatory agents)and siNLRP3+LPS+ATP group(silenced target gene and added proinflammatory agents).The expression levels of NLRP3 and Caspase-1 were detected by Western blot and real-time polymerase chain reaction,and analyzed by analysis of variance for 2-factor factorial design.Results The microvascular segments of rat BMECs were“beaded”after 24 h of isolation and culture;after 48 h,“island”cell clusters were formed;after 72 h,“paving stone”like monolayer cells adhered to the wall and grew.After that,the cells gradually became dense and reached the convergence degree of 80%.The positive rate of BMECs detected by immunofluorescence staining was 96%.In the normally cultured cells,the protein and mRNA expression levels of NLRP3 and Caspase-1 in the LPS+ATP group were higher than those in the blank control group(P<0.05).In the

关 键 词：NOD样受体家族含pyrin结构域蛋白3 沉默基因表达 脑微血管内皮细胞 脑小血管病 

分 类 号：R392[医药卫生—免疫学]