目的 探讨日本血吸虫感染小鼠脾脏滤泡辅助性T（Tfh）细胞的转录组学特征，筛选并验证关键差异基因，旨在揭示Tfh细胞在血吸虫感染免疫应答中的潜在调控作用。方法 选取C57BL/6小鼠随机分为感染组和对照组（各8只），感染组经皮肤接触感染日本血吸虫尾蚴，对照组不作处理。于感染后6周采集脾脏，通过磁珠负选联合流式细胞术依据白细胞分化抗原3、白细胞分化抗原4、程序性死亡受体1、程序性死亡受体1阳性（CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺）的免疫表型设门分选Tfh细胞，RNA提取试剂盒提取细胞样品总RNA并纯化。利用Illumina平台进行Bulk RNA-seq测序，筛选差异表达基因，筛选标准为|log₂差异倍数| > 1且P < 0.05，并进行基因本体论（GO）及京都基因与基因组百科全书（KEGG）富集分析。将筛选出的基因进行逆转录实时荧光定量PCR（RT-qPCR）验证，采用ELISA法检测感染后不同时间点（2、4、6、8周）小鼠血清中白细胞介素5（IL-5）和IL-13的蛋白表达水平。结果 转录组测序共筛选出1 379个差异基因，其中上调基因327个，下调基因1 052个。GO、KEGG功能富集分析显示，差异基因主要富集于细胞因子-细胞因子受体相互作用、趋化性及免疫效应过程调控等信号通路。筛选出il5、il13、前列腺素D2受体2（ptgdr2）、神经介素U受体1（nmur1）等4个差异基因进行RT-qPCR验证，结果显示感染组中il5、il13的mRNA相对转录水平分别为13.520 ± 1.540、22.930 ± 0.720，高于对照组的1.060 ± 0.225、1.137 ± 0.425（t = 10.52、28.41，P < 0.01）；nmur1和ptgdr2在感染组中的相对转录水平分别为11.210 ± 4.480、4.405 ± 2.575，与对照组（1.007 ± 0.084、1.143 ± 0.443）相比差异无统计学意义（t = 3.054、1.629，P > 0.05）。ELISA结果显示，在未感染及感染早期（第2周）时，IL-5和IL-13的浓度均处于极低水平（中位数分别为2.00和4.00 pg/mL），感染后第4周IL-13浓度中位数升至68.20 pg/mL，第6周IL-5浓度中位数升至77.06 pg/mL（均P < 0.05），且在后续感染阶段持续维持较高水平。结论 日本血吸虫感染显著重塑了小鼠脾脏Tfh细胞的转录组特征，使其处于高度活化状态；Tfh细胞可能通过显著上调il5和il13的表达，参与宿主Th2型免疫微环境的维持及免疫病理调控。

Objective To investigate the transcriptomics characteristics of splenic T follicular helper (Tfh) cells in mice infected with Schistosoma japonicum, and to screen and validate key differentially expressed genes (DEGs), so as to unravel the potential regulatory role of Tfh cells in the immune response to S. japonicum infection. Methods C57BL/6 mice were randomly divided into an infection group and a control group, of 8 mice each group. Mice in the infection group were infected with S. japonicum cercariae via skin contact, while animals in the control group received no treatment. Mouse spleens were sampled 6 weeks post-infection, and Tfh cells were isolated by gating based on the immune phenotype of positive cluster of differentiation 3, cluster of differentiation 4, CXC subfamily receptor 5, programmed death-1 (CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺) by means of negative selection with magnetic beads and flow cytometry. Total RNA was extracted from cell samples with an RNA extraction kit and purified. Bulk RNA-seq sequencing was performed on the Illumina platform to screen DEGs [|log₂FoldChange| > 1 and P < 0.05], and DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and screened DEGs were validated using reverse transcription quantitative Real-time PCR (RT-qPCR) assay. The expression of interleukin 5 (IL-5) and IL-13 proteins was measured using enzyme-linked immunosorbent assay (ELISA) in the serum of mice 2, 4, 6 and 8 weeks post-infection. Results Transcriptome sequencing identified a total of 1 379 DEGs, including 327 upregulated and 1 052 downregulated genes. GO and KEGG functional enrichment analyses revealed that these genes were significantly enriched in signal pathways of cytokine-cytokine receptor interaction, chemotaxis, and immune effector process regulation. RT-qPCR assay quantified higher relative il5 [(13.520 ± 1.540) vs. (1.060 ± 0.225); t = 10.52, P < 0.01] and il13 mRNA expression [(22.930 ± 0.720) vs. (1.137 ± 0.425); t = 28.41, P < 0.01] in the infection group than in the control group, and showed no significant difference between the infection and control groups in terms of relative neuromedin U receptor 1 (nmur1) [(11.210 ± 4.480) vs. (1.007 ± 0.084); t = 3.054, P > 0.05] or prostaglandin D2 receptor 2 (ptgdr2) mRNA expression [(4.405 ± 2.575) vs. (1.143 ± 0.443); t = 1.629, P > 0.05]. ELISA measured extremely low IL-5 (median, 2.00 pg/mL) and IL-13 concentrations (median, 4.00 pg/mL) in both uninfected mice and at early stages of infections (2 weeks post-infection). The median IL-13 concentration increased to 68.20 pg/mL at 4 weeks post-infection (P < 0.05), and the median IL-5 concentration rose to 77.06 pg/mL at 6 weeks post-infection (P < 0.05), which was maintained a high level in subsequent infection phases. Conclusion S. japonicum infection remarkably reshapes the transcriptome profile of murine splenic Tfh cells, leading to a highly activation state. Tfh cells may participate in maintaining the host Th2-type immune microenvironment and regulating immunopathology through significantly upregulating il5 and il13 expression.