弓形虫VEG株ROP16蛋白对THP-1细胞免疫调节及机制研究  

Study on immunomodulation of ROP16 protein from VEG strain of Toxoplasma gondii on THP-1 cells and its mechanism

作　　者：陈梅 贾伟 杨峥[1] 党甜甜 潘亚菲 苏雅静 杨宁爱 赵志军 CHEN Mei;JIA Wei;YANG Zheng;DANG Tian-tian;PAN Ya-fei;SU Ya-jing;YANG Ning-ai;ZHAO Zhi-jun(College of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Clinical and Pathogenic Microbiology,Yinchuan 7500043,China;Laboratory Medical Center,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,宁夏银川750004 [2]宁夏临床病原微生物重点实验室,宁夏银川750004 [3]宁夏医科大学总院医学实验中心,宁夏银川750004

出　　处：《中国兽医科学》2022年第4期466-474,共9页Chinese Veterinary Science

基　　金：国家自然科学基金项目(81560333)。

摘　　要：为了探究弓形虫VEG株ROP16蛋白在THP-1细胞中的表达及对其免疫反应的影响和相关机制,本研究利用慢病毒构建过表达ROP16(overEep-ROP16)载体,空载体(overEep-NC)为对照,通过转染THP-1细胞构建稳转细胞株,对其荧光蛋白表达情况、ROP16在THP-1细胞中的表达及定位进行检测;通过RT-PCR检测炎性因子IL-1β、IL-18、TNF-α、IL-10、IL-12 mRNA相对表达水平;Western-blot方法检测炎性小体NLRP3、Caspase-1蛋白与信号通路蛋白STAT3、pTyr705-STAT3的表达水平。结果显示,构建的过表达ROP16重组慢病毒转染THP-1细胞后可观察到强绿色荧光,在THP-1细胞中rop16基因在mRNA与蛋白水平均成功表达且该蛋白定位于THP-1细胞核;与对照组相比,过表达组促炎细胞因子IL-1β、IL-18、TNF-αmRNA表达水平上调(P<0.01),而IL-12 mRNA下调(P<0.01),抗炎细胞因子IL-10 mRNA上调(P<0.01),炎性小体NLRP3、Caspase-1蛋白与信号通路蛋白pTyr705-STAT3相对表达量上调(P<0.01),STAT3无显著变化(P>0.05)。上述结果表明,构建的过表达ROP16重组慢病毒稳转染至THP-1细胞内并表达蛋白,表达的蛋白定位于THP-1细胞核内,该蛋白能激活NLRP3炎性小体促发炎症的发生并通过磷酸化STAT3 Tyr705调控抗炎作用。To investigate the expression of ROP16 protein from VEG strain of Toxoplasma gondii in THP-1 cells and its effect on immune response and related mechanisms,in this study,lentivirus was used to construct overexpression ROP16(over Eep-ROP16)vector and empty vector(over Eep-NC)were used as the control.Stable transfer cell line was constructed by transfecting THP-1 cells.The expression of fluorescence protein,the expression and localization of ROP16 in THP-1 cells were determined.The expression levels of inflammatory cytokines IL-1β,IL-18,TNF-α,IL-10 and IL-12 mRNA was detected by RT-PCR.The expression levels of in flammasome NLRP3,Caspase-1 proteins and signal pathway proteins STAT3,ptyr705-STAT3 were tested by Western-blot.The results showed that strong green fluorescence could be observed after the constructed recombinant lentivirus overexpressing ROP16 was transferred to THP-1cells.Moreover,rop16 gene was successfully expressed at mRNA and protein levels in THP-1 cells and the protein was localized in the nucleus of THP-1 cells.Compared with the control group,the pro-inflammatory cytokines IL-1β,IL-18 and TNF-αmRNA were up-regulated(P<0.01).However,the IL-12 mRNA was down-regulated(P<0.01),and the anti-inflammatory cytokine IL-10 mRNA was up-regulated(P<0.01)in the overexpression group.The relative expressions of NLRP3,Caspase-1 proteins and p Tyr705-STAT3 protein were up-regulated(P<0.01),but STAT3 protein had no significant change(P>0.05)in the overexpression group.This study demon strated that the constructed recombinant lentivirus with overexpression of ROP16 could stably transfer to THP-1 cells and express ROP16 protein,which was located in the nucleus of THP-1 cells.The protein can activate the NLRP3 inflammasome to induce the occurrence of inflammation and regulate anti-inflammatory effects through phosphorylating STAT3 Tyr705.

关 键 词：刚地弓形虫 ROP16蛋白 THP-1细胞 炎性小体 STAT3 

分 类 号：S852.729[农业科学—基础兽医学]