弓形虫Ⅱ型ROP16蛋白对大鼠肺泡巨噬细胞NR8383炎性反应的影响  

Effects of Toxoplasma Gondii TypeⅡROP16 Protein on the Inflammatory Response of Rat Alveolar Macrophages NR8383

作　　者：党甜甜 贾伟[2,3] 陈梅 潘亚菲 杨宁爱 康宇婷 苏雅静 汪澎涛 赵志军 DANG Tiantian;JIA Wei;CHEN Mei;PAN Yafei;YANG Ning'ai;KANG Yuting;SU Yajing;WANG Pengtao;ZHAO Zhijun(College of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Pathogenic Microorganisms,Ningxia Medical University,Yinchuan 750004,China;Medical Experimental Center of General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学病原微生物重点实验室,银川750004 [3]宁夏医科大学总医院医学实验中心,银川750004

出　　处：《宁夏医科大学学报》2022年第4期360-366,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(81560333);2018年宁夏高等学校科学研究项目(NGY2018-103)。

摘　　要：目的探讨弓形虫Ⅱ型ROP16蛋白对大鼠肺泡巨噬细胞NR8383炎性反应的影响。方法实验设置空白对照组(正常NR8383细胞)、空载组(pHBLV-CMV)和ROP16过表达组(pHBLV-CMV ROP16),构建ROP16过表达慢病毒载体并转染至NR8383细胞中,经筛选得到稳转细胞系,利用RT-PCR和Western blot法验证慢病毒在ROP16过表达组是否转染成功;免疫荧光法确定ROP16蛋白在NR8383细胞中的定位;Western blot法检测诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)、核转录因子κB(NF-κB)、磷酸化核转录因子κB(P-NF-κB)、信号转导与转录激活因子1(STAT1)及磷酸化转录活化蛋白1(P-STAT1)蛋白的表达情况;RT-PCR检测iNOS、Arg-1及炎性因子TNF-α、IL-1β、IL-6、TGF-β、IL-10 mRNA水平的表达情况;ELISA检测培养上清中炎性因子TNF-α、IL-1β、IL-6、TGF-β、IL-10的含量。结果ROP16过表达慢病毒成功稳转NR8383细胞,镜下可观察到明显绿色荧光;免疫荧光结果显示,ROP16蛋白定位于NR8383细胞核中;Western blot结果显示,ROP16过表达组中M1型巨噬细胞表面标志蛋白iNOS的表达高于空白对照组,其通路蛋白NF-κB、P-NF-κB、STAT1、P-STAT1的表达均高于空白对照组(P均<0.05),而M2型巨噬细胞表面标志蛋白Arg-1表达低于空白对照组(P<0.05);RT-PCR结果显示,ROP16过表达组iNOS及促炎因子TNF-α、IL-1β、IL-6 mRNA均升高(P均<0.01),而Arg-1及抗炎因子TGF-β、IL-10 mRNA均下降(P均<0.05);ELISA结果显示,过表达组中促炎因子TNF-α、IL-1β、IL-6及IL-12含量均高于空白对照组(P均<0.05),而抗炎因子TGF-β和IL-10的表达均低于空白对照组(P均<0.05)。结论弓形虫Ⅱ型ROP16定位于NR8383巨噬细胞核中并稳定表达,同时向M1型巨噬细胞方向极化,引发促炎反应。Objective To investigate the effect of Toxoplasma gondii typeⅡROP16 protein on the inflammatory response of rat alveolar macrophages NR8383.Methods A blank control group(normal NR8383 cells),a no-load group(pHBLV-CMV)and a ROP16 overexpression group(pHBLV-CMV ROP16)were set up to construct ROP16 overexpression lentiviral vector and transfected into NR8383 cells,after screening,stable cell lines were obtained,the ROP16 overexpression group was successfully transfected and detected by RT-PCR and Western blot;the localization of ROP16 protein in NR8383 cells was determined by immunofluorescence;the expression of iNOS,Arg-1 and NF-κB,P-NF-κB,STAT1 and P-STAT1 protein levels were detected by Western blot;the expression of iNOS,Arg-1 and inflammatory factors TNF-α,IL-1β,IL-6,TGF-β,IL-10 mRNA levels were detected by RT-PCR.ELISA to used to detect the levels of inflammatory factors TNF-α,IL-1β,IL-6,TGF-β,IL-10 in culture supernatant.Results The ROP16 overexpression lentivirus successfully stably transfected NR8383 cells,and obvious green fluorescence could be observed under the microscope;immunofluorescence results showed that ROP16 protein was localized in the nucleus of NR8383 cells;Western blot results showed that the expression of M1-type macrophage surface marker protein iNOS was higher in the ROP16 overexpression group than in the blank control group,and the expression of its pathway proteins NF-κB,P-NF-κB,STAT1,and P-STAT1 in the ROP16 overexpression group were higher than those in the blank control group(P all<0.05),while the expression of Arg-1,a marker protein on the surface of M2 macrophages,was lower than that in the blank control group(P<0.05);RT-PCR results showed that iNOS and pro-inflammatory factors TNF-α,IL-1β,and IL-6 mRNA were increased in the ROP16 overexpression group(P all<0.01),while Arg-1 and anti-inflammatory factors TGF-βand IL-10 mRNA were decreased(P all<0.05);ELISA results showed that the levels of pro-inflammatory factors TNF-α,IL-1β,IL-6 and IL-12 were higher in the overex

关 键 词：Ⅱ型ROP16蛋白 NR8383细胞 巨噬细胞极化 

分 类 号：R392[医药卫生—免疫学]