刚地弓形虫Ⅰ/Ⅲ型ROP16调控TAF15对THP‑1细胞影响及机制研究  

Effect and mechanism of Toxoplasma gondii ROP16Ⅰ/Ⅲregulating TAF15 on THP‑1 cells

作　　者：殷荷 马磊 党甜甜 李佳铭 赵志军 YIN He;MA Lei;DANG Tiantian;LI Jiaming;Zhao Zhijun(General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia,China;Ningxia Medical Laboratory Clinical Research Center,Yinchuan 750004,Ningxia,China;College of Life Sciences of Ningxia University,Yinchuan 750004,Ningxia,China)

机构地区：[1]宁夏医科大学总医院医学实验中心,宁夏银川750004 [2]宁夏医学检验临床医学研究中心,宁夏银川750004 [3]宁夏大学生命科学学院,宁夏银川750004

出　　处：《中国寄生虫学与寄生虫病杂志》2025年第1期103-111,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：宁夏自然科学基金(2024AAC03696);宁夏回族自治区重点研发计划(2023BEG02002)。

摘　　要：目的探究刚地弓形虫Ⅰ、Ⅲ型棒状体蛋白16(ROP16)通过调控TATA结合蛋白相关因子15(TAF15)对人单核细胞白血病细胞(THP-1)增殖、凋亡的影响及作用机制。方法将Ⅰ、Ⅲ型ROP16过表达慢病毒转染至THP-1细胞,构建稳定表达ROP16的细胞株(THP-1-ROP16Ⅰ/Ⅲ),以转染空载体慢病毒的细胞为空载体对照组(THP-1-Venus),未转染细胞为对照组(THP-1),通过实时荧光定量逆转录PCR(RT-qPCR)和蛋白质免疫印迹(Western blotting)验证过表达效果。利用免疫沉淀-质谱联用技术(IP-MS)在THP-1-ROP16Ⅰ/Ⅲ细胞株中预测ROP16的互作蛋白,并通过RT-qPCR和Western blotting检测细胞中互作蛋白TAF15的表达。将3条作用于TAF15基因不同位点的小干扰RNA(siRNA 1215、siRNA 825、siRNA 288)分别干扰THP-1-ROP16Ⅰ/Ⅲ细胞株,分为THP-1-ROP16Ⅰ/Ⅲ+siRNA 1215/825/288组,同时设未干扰对照组(THP-1-ROP16Ⅰ/Ⅲ+siRNA NC),Western blotting验证TAF15沉默效果。通过细胞计数试剂盒8(CCK-8)与流式细胞术检测各组细胞增殖及凋亡情况,Western blotting检测细胞周期依赖性蛋白激酶抑制因子1A(p21)、周期素依赖性激酶6(CDK6)、G1/S特异性周期蛋白(CyclinD1)、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解型半胱氨酸蛋白酶-3(cleaved caspase-3)、半胱氨酸蛋白酶-9(caspase-9)及磷酸化信号转导和转录激活因子3(P-STAT3)蛋白的表达。结果THP-1-ROP16Ⅰ/Ⅲ组ROP16 mRNA的相对转录水平为2679.427±250.600、2395.410±325.700,均高于THP-1-Venus组(1.036±0.102)(F=153.3,P<0.01);ROP16蛋白的相对表达水平为4.526±0.020、5.457±0.250,均高于THP-1-Venus组(1.688±0.653)(F=76.4,P<0.01)。TAF15为Ⅰ、Ⅲ型ROP16的互作蛋白,THP-1-ROP16Ⅰ/Ⅲ组TAF15 mRNA的相对转录水平为6.027±0.313、5.567±0.088,均高于THP-1-Venus组(0.985±0.027)(F=869.4,P<0.01);TAF15蛋白的相对表达水平为1.789±0.145、1.593±0.029,均高于THP-1-Venus组(1.010±0.365)(F=50.6,P<0.01)。TAF15 siRNA转染Objective To investigate the effect and mechanisms of Toxoplasma gondii typeⅠandⅢrhoptry protein 16(ROP16)on the proliferation and apoptosis of human monocytic leukemia THP-1 cells via TATA-binding protein-associated factor 15(TAF15).Methods THP-1 cells were transfected with entiviruses overexpressing T.gon⁃dii typeⅠandⅢROP16 to generate cell lines that stably expressed ROP16(THP-1-ROP16Ⅰ/Ⅲ),and cell trans-fected with lentiviruses containing empty vectors(THP-1-Venus)served as an empty vector control,while non-trans-fected cells(THP-1)served as controls.The efficiency of overexpression was checked using quantitative real-time PCR(RT-qPCR)assay and Western blotting.The proteins interacting with ROP16 were identified using immunoprecipi-tation-mass spectrometry(IP-MS)in THP-1-ROP16Ⅰ/Ⅲcell lines,and the expression of the ROP16-interacting pro-tein TAF15 was quantified using RT-qPCR and Western blotting assays.Three siRNA targeting different sites of TAF15 gene(siRNA 1215,siRNA 825,siRNA 288)were used to interfere with THP-1-ROP16Ⅰ/Ⅲcell lines and divided into THP-1-ROP16Ⅰ/Ⅲ+siRNA 1215/825/288 groups,while an undisturbed control group(THP-1-ROP16Ⅰ/Ⅲ+siRNA NC)was set up and the silencing efficiency of TAF15 was checked using Western blotting.In addition,the cell proliferation and apoptosis was measured using cell counting kit-8(CCK-8)assay and flow cytometry,and the expression of cyclin-dependent kinase inhibitor 1A(p21),cyclin-dependent kinase 6(CDK6),G1/S-specific cyclin(Cy-clinD1),B-cell lymphoma/leukemia-2 protein(Bcl-2),Bcl-2-associated X protein(Bax),cleaved caspase-3,caspase-9 and phosphorylated signal transducer and activator of transcription 3(P-STAT3)was determined using Western blot-ting.Results The relative ROP16 mRNA expression was 2679.427±250.600 in the THP-1-ROP16Ⅰgroup and 2395.410±325.700 in the THP-1-ROP16Ⅲgroup,which was both higher than in the THP-1-Venus group(1.036±0.102)(F=153.3,P<0.01),and the relative ROP16 protein expression was higher in the THP-1-ROP16�

关 键 词：刚地弓形虫 棒状体蛋白16 TATA结合蛋白相关因子15 THP‑1细胞 

分 类 号：R382.5[医药卫生—医学寄生虫学]