目的 探讨Tsc22结构域家族成员3（Tsc22 domain family member 3，Tsc22d3）在日本血吸虫感染小鼠肝脏自然杀伤（natural killer，NK）细胞亚群中的表达及其对NK细胞杀伤功能的影响。方法 将42只C57BL/6 雌性小鼠经腹部贴片法感染日本血吸虫尾蚴（20 ± 1 条/只），分别在感染前、感染后第4、6周取6只小鼠剖杀，磁珠分选富集肝脏NK细胞，并进行单细胞测序，分析Tsc22d3在NK细胞的表达及活化的信号通路。其余24只小鼠随机均分为感染组和对照组，每组12只，感染后第4、6周各取6只小鼠进行麻醉，分离小鼠肝非实质细胞，流式细胞术检测Tsc22d3在对照组和感染组小鼠肝脏NK细胞中的表达变化。采用脂质体转染法将空载质粒、Tsc22d3表达质粒转染NK92细胞，获得空载质粒对照组（NC组）和Tsc22d3过表达NK92细胞组（Tsc22d3⁺NK92细胞组）；用可溶性虫卵抗原（SEA）刺激Tsc22d3⁺NK92组和NC组24 、48 h，流式细胞术和实时荧光定量PCR（qPCR）检测NK细胞中γ干扰素（IFN-γ）和穿孔素（Prf1）表达的变化。使用GraphPad Prism9软件进行统计学分析，两组比较采用t检验。结果 单细胞测序结果提示，与感染前小鼠（2.41）相比，感染后第4周（3.16）、第6周（3.76）Tsc22d3的表达持续增加，Tsc22d3高表达于NK的C3亚群，KEGG结果显示C3亚群中NK细胞介导的细胞毒性通路显著活化；流式细胞术结果显示，感染组第4、第6周小鼠肝脏NK细胞中Tsc22d3⁺NK的占比分别为（0.24 ± 0.01）%、（1.40 ± 0.14）%，高于对照组的（0.12 ± 0.02）%（t = 12.110、22.010，均P < 0.01）。SEA刺激24 h后Tsc22d3⁺NK92组中IFN-γ、Prf1的阳性细胞的占比分别为（1.66 ± 0.15）%、（53.23 ± 0.81）%，高于NC组的（0.88 ± 0.13）%、（29.93 ± 1.85）%（t = 6.800、20.010，均P < 0.01）。SEA刺激24 h后，Tsc22d3⁺NK92组中IFN-γ、Prf1的mRNA相对转录水平分别为1.53 ± 0.24、1.41 ± 0.04，均高于NC组的1.00 ± 0.07、1.00 ± 0.14（t = 3.573、4.973，均P < 0.05、0.01）；SEA刺激48 h后Tsc22d3⁺NK92组中IFN-γ、Prf1的mRNA相对转录水平分别为2.06 ± 0.39、1.54 ± 0.26，均高于NC组的1.06 ± 0.44、1.00 ± 0.04（t = 2.953、3.588，均P < 0.05）。结论 日本血吸虫感染进程中Tsc22d3高表达于肝脏NK的C3亚群，Tsc22d3高表达可增强NK细胞的杀伤功能。

Objective To investigate the expression of Tsc22 domain family member 3 (Tsc22d3) expression in natural killer (NK) cell subsets in liver of mice infected with Schistosoma japonicum and its effect on the cytotoxicity of NK cells. Methods Forty-two female C57BL/6 mice were infected with S. japonicum cercariae (20 ± 1 per mouse) by abdominal patch method. Six mice were sacrificed before infection and at the 4th and 6th weeks after infection respectively. NK cells in the liver were enriched by magnetic bead sorting and single-cell sequencing was performed to analyze the expression of Tsc22d3 in NK cells and the activated signaling pathways. The remaining 24 mice were randomly divided into an infection group and a control group, with 12 mice in each group. At the 4th and 6th weeks after infection, 6 mice in each group were anesthetized and non-parenchymal cells in the liver were isolated. The Tsc22d3 expression was detected in mouse hepatic NK cells in the control and infection groups using flow cytometry. Empty plasmid and Tsc22d3 plasmid were transfected into NK92 cells to obtain the empty plasmid control group (NC group) and the Tsc22d3 overexpression NK92 cell group (Tsc22d3⁺NK92 group) by using liposome transfection method. NK92 cells in the Tsc22d3⁺NK92 group and the NC group were stimulated with soluble egg antigen (SEA) for 24 or 48 h, and the expression of interferon-γ (IFN-γ) and perforin 1 (Prf1) was detected in NK cells using flow cytometry and real-time quantitative fluorescence PCR (qPCR) assay. All statistical analyses were performed with the software GraphPad Prism 9, and differences of means between the two groups was tested for statistical significance with t test. Results Single-cell sequencing revealed that compared with uninfected mice (2.41), the expression of Tsc22d3 continued to increase at 4 weeks (3.16) and 6 weeks after infection (3.76), and Tsc22d3 was highly expressed in the C3 subset of NK cells. The pathway of natural killer cell mediated cytotoxicity in the C3 subgroup were significantly activated. Flow cytometry showed that the proportions of Tsc22d3⁺NK92 cells were (0.24 ± 0.01)% and (1.40 ± 0.14)% in mouse hepatic NK cells 4 weeks and 6 weeks post-infection in the infection group, which were both higher than in the control group (0.12% ± 0.02%) (t = 12.110 and 22.010, both P values < 0.01). The proportions of IFN-γ and Prf1 positive cells were (1.66 ± 0.15)% and (53.23 ± 0.81)% in the Tsc22d3⁺NK92 group 24 h post-stimulation with SEA, which higher than in the NC group [(0.88 ± 0.13)% and (29.93 ± 1.85)%, respectively] (t = 6.800 and 20.010, both P values < 0.01), and the relative mRNA expression of IFN-γ and Prf1 was (1.53 ± 0.24) and (1.41 ± 0.04) in the Tsc22d3⁺NK92 group 24 h post-stimulation with SEA, which was higher than in the NC group [(1.00 ± 0.07) and (1.00 ± 0.140), respectively] (t = 3.573 and 4.973, P < 0.05 and 0.01). The relative mRNA expression of IFN-γ and Prf1 was (2.06 ± 0.39) and (1.54 ± 0.26) in the Tsc22d3⁺NK92 group 48 h post-stimulation with SEA, which was higher than in the NC group [(1.06 ± 0.44) and (1.00 ± 0.04), respectively] (t = 2.953 and 3.588, both P values < 0.05). Conclusion Tsc22d3 is highly expressed in the C3 subset of hepatic NK cells during the course of S. japonicum infection, and high Tsc22d3 expression may increase the cytotoxicity of NK cells.