弓形虫RH株ROP16 I蛋白通过JAK-STAT3途径对MH-S细胞增殖及细胞周期影响的研究  

Toxoplasma gondii RH strain ROP16 I protein affects proliferation and the cell cycle in MH-S cells through the JAK-STAT3 pathway

作　　者：李佳铭 党甜甜 殷荷 赵志军 LI Jia-ming;DANG Tian-tian;YIN He;ZHAO Zhi-jun(Medical Experimental Center,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Pathogenic Microorganisms,Yinchuan 750004,China;Ningxia Clinical Research Center of Medical Laboratory,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院医学实验中心,银川750004 [2]宁夏病原微生物重点实验室,银川750004 [3]宁夏医学检验临床研究中心,银川750004

出　　处：《中国人兽共患病学报》2025年第2期113-120,共8页Chinese Journal of Zoonoses

基　　金：宁夏回族自治区重点研发计划项目(No.2023BEG02002);宁医大总院2023年新入职硕士人才培养项目(No.〔2023〕394)联合资助。

摘　　要：目的 探究I型弓形虫RH株ROP16蛋白对小鼠肺泡巨噬细胞株MH-S细胞增殖及细胞周期的影响和机制。方法 将ROP16 I过表达慢病毒感染MH-S细胞后经嘌呤霉素筛选获得稳定表达ROP16 I的过表达细胞株。RT-qPCR和Western-blot验证过表达效果,cck-8检测细胞增殖活性,流式细胞术检测细胞周期变化。Western-blot及RT-qPCR检测p53、p21、CDK6、Cyclin D1、STAT3、p-STAT3(Y705)及JAK1蛋白或基因的表达水平,免疫荧光检测ROP16 I与p-STAT3(Y705)在MH-S细胞中的亚细胞共定位。结果 ROP16 I过表达慢病毒感染MH-S细胞后可检测到ROP16 I蛋白及基因表达。cck-8检测发现ROP16 I可促进MH-S细胞增殖(P<0.01),提升细胞活力。流式细胞术检测发现ROP16 I过表达可使MH-S细胞细胞周期G0/G1期降低,G2期、S期升高。相较于MH-S细胞组、MH-S-vector组,MH-S-ROP16细胞组p53及p21蛋白表达降低,CDK6、Cyclin D1、p-STAT3(Y705)及JAK1蛋白表达升高,p53及p21 mRNA表达降低,CDK6及Cyclin D1 mRNA表达升高(均P<0.01)。免疫荧光检测发现ROP16 I与p-STAT3(Y705)共同定位于细胞核及其周围细胞质中。结论 I型弓形虫RH株ROP16蛋白可激活JAK-STAT3途径,诱导细胞周期G0/G1期缩短,G2/S期延长,促进细胞增殖,为揭示弓形虫的免疫逃避机制提供了理论依据,为弓形虫肺炎防治研究奠定了理论基础。This study was aimed at investigating the effects and mechanisms of Toxoplasma gondii type I(RH strain)ROP16 protein on proliferation and the cell cycle in mouse alveolar macrophage MH-S cells.We constructed a Toxoplasma gondii type I(RH)ROP16 overexpression lentivirus,transduced MH-S cells,and then screened cells with puromycin to obtain a cell line stably overexpressing ROP16 I.RT-qPCR and western blotting were used to verify expression effects,CCK-8 assays were used to detect cell proliferation activity,and flow cytometry was used to detect cell cycle changes.Western blotting and RT-qPCR were used to detect the expression levels of p53,p21,CDK6,Cyclin D1,STAT3,p-STAT3(Y705),and JAK1 proteins or genes,and immunofluorescence was used to detect the expression levels of ROP16 I and p-STAT3(Y705)and their subcellular co-localization in MH-S cells.ROP16 I protein and gene expression were detected in MH-S cells transduced with lentivirus for ROP16 I overexpression.CCK-8 assays revealed that ROP16 I promoted the proliferation of MH-S cells(P<0.01)and enhanced cell viability.Flow cytometry revealed that ROP16 I overexpression decreased the G0/G1 phase and elevated the G2 and S phases of the cell cycle in MH-S cells(P<0.01 or P<0.05).Compared with the MH-S cell group and MH-S-empty vector group,the MH-S-ROP16 cell group showed lower expression of p53 and p21 proteins;higher expression of CDK6,Cyclin D1,p-STAT3(Y705),and JAK1 proteins;lower expression of p53 and p21 mRNAs;and higher expression of CDK6 and Cyclin D1 mRNAs(all P<0.01).Immunofluorescence revealed that ROP16 I co-localized with p-STAT3(Y705)in the nucleus and surrounding cytoplasm.Therefore,Toxoplasma gondii type I(RH)ROP16 I protein activates the JAK-STAT3 pathway;shortens the G0/G1 phase and lengthens the G2/S phase of the cell cycle;and promotes cell proliferation.These findings provide a theoretical basis for revealing the mechanism of immune evasion of Toxoplasma gondii,and lay a foundation for research on the prevention and treatment of Toxoplasma gond

关 键 词：ROP16蛋白 MH-S细胞 细胞周期 STAT3 p-STAT3(Y705) 

分 类 号：R382.5[医药卫生—医学寄生虫学]