摘要
目的探究弓形虫Ⅰ型ROP16蛋白对THP-1细胞炎性反应的影响及相关机制。方法构建过表达ROP16(pHBLV-CMV ROP16)和空载体(pHBLV-CMV)慢病毒,转染THP-1细胞,建立稳定过表达ROP16细胞组、空载体及未转染的空白对照,转染72 h后荧光显微镜下观察绿色荧光表达情况,采用RT-PCR与Western blot检测ROP16 mRNA及蛋白在THP-1细胞中的表达,验证转染情况;稳转细胞采用RT-PCR检测炎性因子白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)、白细胞介素-10(IL-10)、白细胞介素-12(IL-12)mRNA表达水平;通过免疫荧光法检测ROP16蛋白在THP-1细胞中的定位;采用Western blot检测THP-1细胞中炎性小体NLRP3、Caspase-1蛋白的表达水平。结果构建的过表达ROP16重组慢病毒成功稳转THP-1细胞,可观察到表达的绿色荧光蛋白;免疫荧光法检测ROP16蛋白定位于THP-1细胞核;pHBLV-CMV ROP16过表达慢病毒载体转染THP-1细胞后ROP16mRNA表达水平较未转染组上调(t=43.63,P<0.01),促炎细胞因子IL-1β、IL-18及炎性小体NLRP3 mRNA表达水平上调(t值分别为12.91、25.61、30.26,均P<0.05),而IL-12 mRNA下调(t=42.35,P<0.01),抗炎细胞因子IL-10 mRNA上调(t=91.39,P<0.05);Western blot显示,pHBLV-CMV ROP16过表达组ROP16蛋白相对表达量高于空白组和空载体组(t值分别为15.67、15.13,P<0.05),炎性小体NLRP3与Caspase-1蛋白相对表达量高于空载体组和空白组(t=22.22、11.29,P<0.05)。结论构建的过表达ROP16重组慢病毒可在THP-1细胞内稳定表达ROP16蛋白,且定位于THP-1细胞核。该蛋白通过调节NLRP3炎性小体进一步促进细胞炎性反应的发生。
Objective To investigate the effect of rhoptry protein 16(ROP16)from theⅠstrain of Toxoplasma gondii on the inflammatory reaction of THP-1 cells and to study its related mechanisms.Methods A lentivirus with overexpression of ROP16(pHBLV-CMV ROP16)and an empty vector(pHBLV-CMV)was constructed.The lentivirus was transfected into THP-1 cells to create a group of cells stably overexpressing ROP16,an empty vector group,and an untransfected blank group.After 72 hours of transfection,the expression of green fluorescence was observed using a fluorescence microscope.The expression of ROP16 mRNA and protein in THP-1 cells was determined using RT-PCR and Western blotting to verify whether transfection was successful.The levels of mRNA expression of the inflammatory factors IL-1β,IL-18,IL-10,and IL-12 were determined using RT-PCR.Immunofluorescence was used to determine the localization of the ROP16 protein in THP-1 cells.The levels of protein expression of the inflammasomes NLRP3 and Caspase-1 in THP-1 cells were determined using Western blotting.Results Results indicated that the recombinant lentivirus overexpressing ROP16 was successfully constructed and transfected into THP-1 cells,and the expression of green fluorescent protein was observed.The ROP16 protein was localized to the nucleus of THP-1 cells.The level of ROP16 mRNA increased after the overexpression vector was transfected into THP-1 cells using pHBLV-CMV ROP16(t=43.63,P<0.01).The levels of mRNA of the pro-inflammatory factors IL-1βand IL-18 and the inflammasome NLRP3 in the pHBLV-CMV ROP16 transfection group were significantly higher than those in the control group(t=12.91,25.61,30.26,P<0.05);the level of mRNA of the anti-inflammatory factor IL-10 increased markedly(t=91.39,P<0.05)while the level of IL-12 mRNA decreased dramatically(t=42.35,P<0.05).Western blot analysis indicated that the relative expression of the ROP16 protein and proteins of the inflammasomes NLRP3 and Caspase-1 increased in the pHBLV-CMV ROP16 transfection group compared to that in the black groups(t=15.67,22.22,11.29,P<0.05).Conclusion A recombinant lentivirus overexpressing ROP16 stably expressed the ROP16 protein in THP-1 cells,and the protein was localized to the nucleus of THP-1 cells.The ROP16 protein further promotes a cellular inflammatory reaction by regulating the inflammasome NLRP3.
作者
陈梅
贾伟
党甜甜
潘亚菲
杨宁爱
苏雅静
康宇婷
汪澎涛
赵志军
CHEN Mei;JIA Wei;DANG Tian-tian;PAN Ya-fei;YANG Ning-ai;SU Ya-jing;KANG Yu-ting;WANG Peng-tao;ZHAO Zhi-jun(College of Clinical Medicine,Ningxia Medical University,Yin-chuan 750004,China;Ningxia Key Laboratory of Clinical and Pathogenic Microbiology;Laboratory Medical Center,General Hospital of Ningxia Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2021年第12期1420-1424,1453,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81560333)
2018年宁夏高等学校科学研究项目(No.NGY2018-103)。
