目的 探讨多房棘球蚴感染早期对小鼠肝组织自然杀伤T（NKT）细胞数量、亚群及其功能的影响。方法 C57BL/6N小鼠随机分为对照组和感染组。感染组小鼠经肝门静脉注射2 000个多房棘球蚴原头节，对照组注射等量生理盐水。感染后4周，取小鼠肝组织，苏木素-伊红（HE）染色观察肝组织病理变化，免疫荧光染色观察肝组织NKT细胞变化。取肝组织制备淋巴细胞悬液，流式细胞术分析对照组和感染组小鼠肝组织NKT细胞及其不同亚群百分比，并检测NKT细胞及其不同亚群中抑制性受体[自然杀伤细胞凝集素样受体2族成员A（NKG2A）和程序性死亡受体（PD-1）]、激活性受体（NKG2D）、趋化因子受体[C-X-C基序趋化因子受体3（CXCR3）和CXCR6]、干扰素-γ（IFN-γ）、肿瘤坏死因子α（TNF-α）、白细胞介素-10（IL-10）和转化生长因子β1（TGF-β1）的表达变化。结果 感染多房棘球蚴后4周，HE染色显示感染组小鼠肝组织内可见具有生发层结构的病灶，周围聚集大量的炎性细胞。免疫荧光染色显示，感染组小鼠肝组织每个视野的平均NKT细胞数为（1.20 ± 0.53）个，低于对照组的（3.73 ± 0.50）个（t = 6.01，P < 0.01）。流式细胞术结果显示，感染组小鼠肝组织NKT细胞、CD69⁺NKT细胞的占比分别为（11.83 ± 1.19）%、（76.93 ± 3.06）%，均低于对照组的（23.20 ± 0.20）%、（88.47 ± 1.53）%（t = 16.28、5.84，均P < 0.01）。感染组分泌IFN-γ和IL-10的肝组织NKT细胞的比例分别为（17.50 ± 5.04）%和（4.54 ± 0.20）%，均低于对照组的（61.90 ± 2.25）%和（10.24 ± 1.37）%（t = 13.93、7.15，P < 0.01、0.05）；感染组分泌TGF-β1的肝组织NKT细胞的比例为9.04%（8.01%, 9.62%），高于对照组的3.68%（3.48%, 3.68%）（t = 8.60，P < 0.05）；感染组分泌TNF-α的肝组织NKT细胞的比例为（20.83 ± 5.75）%，与对照组的（19.20 ± 5.91）%相比差异无统计学意义（t = 0.34，P > 0.05）。感染组肝组织NKG2A⁺NKT、CXCR3⁺NKT和CXCR6⁺NKT细胞的百分比分别为（13.90 ± 3.18）%、（26.77 ± 6.00）%、（57.20 ± 7.63）%，均低于对照组的（22.37 ± 2.61）%、（55.40 ± 13.64）%、（86.73 ± 2.72）%（t = 3.57、3.33、6.31，P < 0.05、0.05、0.01）；感染组肝组织PD-1⁺NKT细胞的百分比为（10.04 ± 1.01）%，高于对照组的（6.56 ± 0.10）%（t = 5.92，P < 0.05）；感染组肝组织NKG2D⁺NKT细胞的百分比为1.58%（1.56%，2.14%），与对照组的1.25%（1.15%，1.31%）（Z = -1.96，P > 0.05）相比差异无统计学意义。感染组肝组织CD4⁺NKT细胞、CD69⁺CD4⁺NKT细胞的比例分别为（42.67 ± 4.15）%、（44.80 ± 2.27）%，均低于对照组的（60.97 ± 1.80）%、（62.30 ± 3.16）%（t = 7.01、7.79，均P < 0.01）。感染组肝组织CD4^-CD8^-（双阴性，DN）NKT细胞、CD69⁺DN NKT细胞的比例分别为（45.63 ± 3.61）%、（44.53 ± 2.23）%，均高于对照组的（32.57 ± 2.06）%、（32.17 ± 1.34）%（t = 5.44、8.23，均P < 0.01）。感染组肝组织分泌IL-10的CD4⁺NKT细胞、分泌IFN-γ和TNF-α的DN NKT细胞占比分别为（9.97 ± 4.38）%、（15.87 ± 2.57）%、（6.61 ± 2.69）%，均低于对照组的（52.00 ± 4.20）%、（59.80 ± 4.70）%、（30.57 ± 2.02）%（t = 11.99、14.20、12.33，均P < 0.01）。感染组肝组织分泌IL-10和TGF-β1的CD8⁺NKT细胞占比分别为（55.90 ± 13.68）%和（45.87 ± 5.95）%，均高于对照组的（10.10 ± 3.24）%和（19.69 ± 10.25）%（t = 5.64、3.83，P < 0.01、0.05）。感染组NKG2A⁺CD4⁺NKT和CXCR3⁺CD4⁺NKT细胞的占比分别为（37.87 ± 4.80）%和（40.40 ± 9.48）%，均低于对照组的（52.80 ± 5.17）%和（57.00 ± 3.90）%（t = 3.66、2.81，均P < 0.05）。感染组NKG2A⁺DN NKT细胞的占比为（55.63 ± 3.46）%，高于对照组的（41.43 ± 2.31）%（t = 5.91，P < 0.01）。结论 在多房棘球蚴感染早期，小鼠肝组织中NKT细胞整体呈功能抑制或耗竭状态；其亚群中，CD4⁺NKT细胞低表达，CD8⁺NKT细胞通过分泌IL-10和TGF-β1发挥作用，高比例活化的DN NKT细胞则因NKG2A的高表达导致其细胞因子分泌能力受损。

Objective To examine the effects of early Echinococcus multilocularis infection on the quantity, subsets, and functions of natural killer T (NKT) cells in mouse hepatic tissues. Methdos C57BL/6N mice were randomly divided into a control group and an infection group. Mice in the infection group were inoculated with 2 000 E. multilocularis protoscoleces via the hepatic portal vein, while animals in the control group received an equal volume of physiological saline. Mouse liver tissues were collected 4 weeks post-infection. The pathological changes of liver tissues were observed by hematoxylin-eosin (HE) staining, and alterations in hepatic NKT cells were examined using immunofluorescence staining. Lymphocyte suspensions were prepared from mouse liver tissues. The percentages of NKT cells and their different subsets were quantified in mouse liver tissues in both control and infection groups using flow cytometry, and the expression of inhibitory receptors [natural killer group 2 member A （NKG2A） and programmed cell death protein 1 (PD-1)], an activating receptor NKG2D, chemokine receptors [C-X-C motif chemokine receptor 3 (CXCR3) and CXCR6], interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), and transforming growth factor-β1 (TGF-β1) was detected in NKT cells and their respective subsets. Results HE staining revealed lesions exhibiting a germinal layer structure in mouse liver tissues in the infection group 4 weeks post-infection with E. multilocularis, surrounded by large number of inflammatory cells. Immunofluorescence staining showed that the average count of NKT cells was (1.20 ± 0.53) cells per field in the liver tissues of the infected group, which was significantly lower than that in the control group (3.73 ± 0.50) cells (t = 6.01, P < 0.01). Flow cytometry detected that the proportions of NKT cells and CD69⁺NKT cells were (11.83 ± 1.19)% and (76.93 ± 3.06)% in mouse liver tissues in the infection group, which were lower than those [(23.20 ± 0.20)% and (88.47 ± 1.53)%, respectively] in the control group (t = 16.28 and 5.84; both P < 0.01). The proportions of hepatic NKT cells secreting IFN-γ and IL-10 were (17.50 ± 5.04)% and (4.54 ± 0.20)% in the infection group, which were lower than those [(61.90 ± 2.25)% and (10.24 ± 1.37)%, respectively] in the control group (t = 13.93, 7.15, P < 0.01, 0.05). The proportion of hepatic NKT cells secreting TGF-β1 was higher in the infected group [9.04% (8.01%, 9.62%)] than in the control group [3.68% (3.48%, 3.68%)] (t = 8.60, P < 0.05), and there was no significant difference in the proportion of hepatic NKT cells producing TNF-α between the infection group [(20.83 ± 5.75)%] and the control group [(19.20 ± 5.91)%] (t = 0.34, P > 0.05). The proportions of NKG2A⁺NKT [(13.90 ± 3.18)% vs. (22.37 ± 2.61)%; t = 3.57, P < 0.05], CXCR3⁺NKT [(26.77 ± 6.00)% vs. (55.40 ± 13.64)%; t = 3.33, P < 0.05] and CXCR6⁺NKT cells [(57.20 ± 7.63)% vs. (86.73 ± 2.72)%; t = 6.31, P < 0.01] were significantly lower in mouse liver tissues in the infection group than in the control group. The percentage of PD-1⁺NKT cells [(10.04 ± 1.01)% vs. (6.56 ± 0.10)%; t = 5.92, P < 0.05] was significantly higher in mouse liver tissues in the infection group than in the control group, and there was no significant difference in the proportion of NKG2D⁺NKT cells in mouse liver tissues between the infection group and the control group [1.58% (1.56%, 2.14%) vs. 1.25% (1.15%, 1.31%); Z = ‒1.96, P > 0.05]. The proportions of CD4⁺NKT cells [(42.67 ± 4.15)% vs. (60.97 ± 1.80)%; t = 7.01, P < 0.01] and CD69⁺CD4⁺NKT cells [(44.80 ± 2.27)% vs. (62.30 ± 3.16)%; t = 7.79, PP < 0.01] were significantly lower in mouse liver tissues in the infection group than in the control group, and the proportions of CD4⁻CD8⁻ (double-negative, DN) NKT cells [(45.63 ± 3.61)% vs. (32.57 ± 2.06)%; t = 5.44, P < 0.01] and CD69⁺DN NKT cells [(44.53 ± 2.23)% vs. (32.17 ± 1.34)%; t = 8.23, P < 0.01] were higher in mouse liver tissues in the infection group than in the control group. The proportions of CD4⁺NKT cells secreting IL-10 [(9.97 ± 4.38)% vs. (52.00 ± 4.20)%; t = 11.99, P < 0.01], and DN NKT cells producing IFN-γ [(15.87 ± 2.57)% vs. (59.80 ± 4.70)%; t = 14.20, P < 0.01] and TNF-α [(6.61 ± 2.69)% vs. (30.57 ± 2.02)%; t = 12.33, P < 0.01] were lower in mouse hepatic tissues in the infection group than in the control group, and the proportions of CD8⁺NKT cells secreting IL-10 [(55.90 ± 13.68)% vs. (10.10 ± 3.24)%; t = 5.64, P < 0.01] and TGF-β1 [(45.87 ± 5.95)% vs. (19.69 ± 10.25)%; t = 3.83, P < 0.05] were significantly higher in mouse liver tissues in the infection group than in the control group. The proportions of NKG2A⁺CD4⁺NKT cells [(37.87 ± 4.80)% vs. (52.80 ± 5.17)%; t = 3.66, P < 0.05] and CXCR3⁺CD4⁺NKT cells [(40.40 ± 9.48)% vs. (57.00 ± 3.90)%; t = 2.81, P < 0.05] were significantly lower in the infection group than in the control group, and the proportion of NKG2A⁺DN NKT cells was higher in the infection group [(55.63 ± 3.46)%] than in the control group [(41.43 ± 2.31)%] (t = 5.91, P < 0.01). Conclusion During the early stage of E. multilocularis infection, NKT cells exhibite an overall state of functional suppression or exhaustion in mouse liver tissues. The NKT cell subsets demonstrate distinct expression characteristics: CD4⁺NKT cells show low expression, and CD8⁺NKT cells function by secreting IL-10 and TGF-β1, while the highly activated DN NKT cells have an impaired cytokine-secreting capacity due to high expression of NKG2A.