目的 转录组测序分析Ⅰ型RH株刚地弓形虫棒状体蛋白16（ROP16）过表达人单核THP-1细胞的基因表达谱变化，筛选免疫应答相关驱动基因。 方法 将人单核THP-1细胞接种于96孔板，过表达组中加入ROP16过表达慢病毒，对照组加入等量培养基，72 h后在荧光显微镜下观察转染情况。TRIzol法提取过表达组和对照组人单核THP-1细胞总RNA，并进行转录组测序，筛选差异表达基因（DEG），绘制火山图。对DEG进行聚类分析、京都基因与基因组百科全书（KEGG）代谢通路富集分析和基因本体（GO）功能富集分类，筛选出过表达组和对照组的DEG，并进行实时荧光定量PCR（qPCR）验证。两组间比较采用独立样本t检验。 结果 荧光显微镜下过表达组90%以上的人单核THP-1细胞表达绿色荧光，对照组细胞未见绿色荧光。qPCR结果表明，过表达组ROP16 mRNA相对转录水平（1 083.484 ± 68.990）较对照组（1.000 ± 0）显著上调（t = 22.9，P < 0.01），表明已构建稳定表达ROP16的THP-1细胞株。共筛选出312个DEG，其中193个表达上调，119个表达下调。KEGG注释结果显示，注释到有机体系统的DEG占比最高（24.2%），显著富集于85个条目，其中免疫系统显著富集的基因有47个。KEGG富集分析发现，DEG富集至20条信号通路上，Th1和Th2细胞分化、自然杀伤细胞介导的细胞毒性及IL-17信号通路等3条与免疫系统相关的通路分别富集DEG 6、7和8个。GO功能注释结果显示，共有309个DEG注释到生物过程、细胞组分和分子功能等3个一级节点分类下的55个二级节点分类中。GO富集结果显示，DEG显著富集于炎症反应、肿瘤坏死因子产物负调控、细胞因子产生、γ干扰素产生的正调节，以及与免疫应答相关的通路中。qPCR检测发现ROP16过表达组中MAN2B1、FOS、C1QA的mRNA相对转录水平（25.994 ± 0.382、60.584 ± 2.968、 36.759 ± 0.180）均高于对照组（1.000 ± 0.039、1.000 ± 0.015、1.000 ± 0）（t = 92.00、28.39、280.7，P < 0.05或0.01），LMNB1、IL-6和 IL-12 mRNA相对转录水平（0.728 ± 0.054、0.517 ± 0.073、0.587 ± 0.015）均低于对照组（1.052 ± 0.027、1.000 ± 0.039、1.000 ± 0.010）（t = 7.64、8.24、33.62，P < 0.05或0.01）。 结论 Ⅰ型RH株弓形虫效应分子ROP16过表达后，人单核THP-1细胞基因表达谱发生了明显变化。

Objective To investigate the changes in the gene expression profile of human monocyte THP-1 cells overexpressing Toxoplasma gondii rhoptry protein 16 (ROP16) using transcriptome sequencing, and to screen immune response-related driver genes. Methods Human monocytic THP-1 cells were seeded onto 96-well plates. Cells in the overexpression group was transfected with ROP16 overexpression lentivirus for 72 h, and cells in the control group were treated with an equal volume of culture medium. The efficiency of cell transfection was checked under a fluorescence microscope. Total RNA was extracted from THP-1 cells and THP-1 cells stably expressing ROP16 using the TRIzol reagent, followed by transcriptome sequencing. Differentially expressed genes (DEG) were screened, and a volcano plot was generated. DEGs were subjected to cluster analysis, Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway enrichment analysis, and gene ontology (GO) functional enrichment classification to screen immune response-related driver genes in human monocytic THP-1 cells following T. gondii infections, and gene expression was quantified using qPCR assay. Differences of means between groups were tested for statistical significance with independent sample t-test. Results Fluorescence microscopy displayed green fluorescence in more than 90% of the cells in the overexpression group in each field of view, and no green fluorescence was observed in the control group. qPCR assay quantified a higher relative expression level of ROP16 mRNA in the overexpression group (1 083.484 ± 68.990) than in the control group (1.000 ± 0) (t = 22.9, P < 0.01), indicating the successful generation of THP-1 cells that stably expressed ROP16. A total of 312 DEGs were identified in THP-1 cells stably expressing ROP16, including 193 upregulated genes and 119 downregulated genes. KEGG annotations showed that the highest proportion of DEGs were annotated to the organism system (24.2%), with 85 items significantly enriched, among which 47 genes were significantly enriched in the immune system. KEGG enrichment analysis showed that DEGs were significantly enriched in 20 signaling pathways, and 6, 7, and 8 DEG were significantly enriched in three pathways related to the immune system, including Th1 and Th2 cell differentiation, natural killer cell-mediated cytotoxicity, and IL-17 signaling pathways, respectively. GO functional annotations showed that a total of 309 DEGs were annotated to 55 secondary node classifications under three primary node classifications of biological processes, cellular components, and molecular functions. GO enrichment analysis showed that DEGs were significantly enriched in inflammatory response, negative regulation of tumor necrosis factor products, cytokine production, positive regulation of interferon-γ production, and immune response-related pathways. RT-qPCR assay detected that higher relative expression of MAN2B1, FOS, C1QA mRNA (25.994 ± 0.382、60.584 ± 2.968、 36.759 ± 0.180) in cells in the ROP16 overexpression group than in the control group (1.000 ± 0.039、1.000 ± 0.015、1.000 ± 0) (t = 92.00, 28.39, 280.7, P < 0.05 or 0.01), and lower relative expression of LMNB1, IL-6, and IL-12 mRNA (0.728 ± 0.054, 0.517 ± 0.073, 0.587 ± 0.015) in the overexpression group than in the control group (1.052 ± 0.027、1.000 ± 0.039、1.000 ± 0.010) (t = 7.64, 8.24, 33.62, P < 0.05 or 0.01). Conclusion The gene expression profile of human monocyte THP-1 cells changes significantly and may play important roles in the immune response following T. gondii infection.