目的 探讨多房棘球蚴感染对小鼠脾自然杀伤T细胞（NKT）及其亚群的影响。 方法 24只C57BL/6小鼠随机分为低、中、高感染组和对照组,每组6只。低、中、高感染组每鼠分别经肝门静脉注射50、500、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2周,无菌条件下取各组小鼠肝脏,制备肝组织切片,常规Masson染色后观察多房棘球蚴病灶和肝纤维化情况;取脾组织制备淋巴细胞悬液,流式细胞术检测NKT细胞不同亚群及分泌细胞因子的水平。结果 Masson染色结果显示,感染2周后,对照组小鼠肝无明显变化;低、中感染组小鼠肝组织仅可见少量肉芽肿,炎性反应较弱,向纤维化修复转变;高感染组肝病灶形成较小的生发层结构,周围炎性细胞分布密集,可见纤维结缔增生。中、高感染组脾NKT细胞绝对数分别为（6.32 ± 0.53）× 10 ⁵、（7.37 ± 1.72）× 10 ⁵,高于对照组的（5.02 ± 1.08）× 10 ⁵和低感染组的（4.90 ± 1.27）× 10 ⁵（P < 0.01）。中、高感染组分泌γ干扰素（IFN-γ）的脾NKT1型细胞比例分别为（13.93 ± 1.61）%、（10.9 ± 3.04）%,低于对照组的（17.3 ± 3.18）%和低感染组的（19.42 ± 2.82）%（ P < 0.01）;低、中、高感染组分泌IL-4的脾NKT2型细胞比例分别为（5.62 ± 0.58）%、（5.86 ± 1.43）%和（5.92 ± 0.94）%,高于对照组的（4.37 ± 0.83）%（ P < 0.05）;低、中感染组分泌IL-17A的脾NKT17型细胞比例分别为（7.02 ± 1.19）%和（6.62 ± 0.41）%,高于对照组的（4.68 ± 0.73）%和高感染组的（3.73 ± 1.04）%（ P < 0.01）。中、高感染组脾CD4 ⁺ NKT 细胞绝对数分别为（1.75 ± 0.36）× 10 ⁵、（1.82 ± 0.24）× 10 ⁵,高于对照组的（1.24 ± 0.26）× 10 ⁵（P < 0.05）;高感染组脾DN NKT细胞绝对数为（2.15 ± 0.78）× 10 ⁵,分别高于对照组的（1.27 ± 0.36）× 10 ⁵、低感染组的（1.20 ± 0.49）× 10 ⁵和中感染组的（1.43 ± 0.19）× 10 ⁵（P < 0.01）。低、中、高感染组分泌IFN-γ的脾CD4 ⁺ NKT1型细胞比例分别为（19.42 ± 2.82）%、（14.40 ± 1.81）%、（12.5 ± 2.96）%,低于对照组的（21.16 ± 2.83）%（ P < 0.01）;低、中、高感染组分泌IL-17A的脾CD4 ⁺ NKT17型细胞比例分别为（18.73 ± 1.99）%、（16.06 ± 1.42）%和（14.41 ± 3.55）%,高于对照组的（10.27 ± 1.79）%（ P < 0.01）。低、中、高感染组分泌IFN-γ的CD8 ⁺ NKT细胞比例分别为（10.04 ± 0.92）%、（13.43 ± 0.92）%和（9.24 ± 1.37）%,低于对照组的（15.83 ± 1.59）%（ P < 0.01）;中、高感染组分泌IL-10的脾CD8 ⁺ NKT细胞比例分别为（4.07 ± 0.48）%、（3.92 ± 0.75）%,高于对照组的（2.85 ± 0.64）%（ P < 0.05）。中、高感染组分泌IFN-γ的脾DN NKT细胞比例分别为（17.48 ± 2.50）%和（14.06 ± 3.95）%,低于对照组的（25.18 ± 5.78）%（ P < 0.01）。 结论 小鼠感染多房棘球蚴后,脾NKT细胞及其亚群发生明显变化,其中低、中感染组小鼠以CD4⁺ NKT17型免疫应答优势对虫体起杀伤和清除作用,高感染组小鼠以CD4⁺ NKT17型、NKT2型和NKT10型细胞亚群的混合优势,且NKT细胞呈抑制性表型,使NKT1型免疫应答能力下降,利于棘球蚴寄生。

Objective To understand the affects of Echinococcus multilocularismetacestode infection to natural killer T (NKT) cells and their subsets in mouse spleen. Methods Twenty-four C57BL/6 mice were randomly assigned into low, medium, high infection groups and control group (6 mice in each). The mice in low, medium, high infection groups were infected with 50, 500, 2 000 protoscoleces, respectively, through hepatic portal vein, while the mice in the control group injected with the same amount of saline. Two weeks after infection, the mice liver and spleen were collected to prepare liver tissue slices. The slices were stained with Masson routine method to observe the metacestodes lesion and liver fibrosis. Spleen lymphocyte suspensions were prepared for examining different NKT subsets, and the secreted cytokines by using flow cytometry.Results Two weeks after infection, there was no significant change in the mice liver of the control group. In the low and medium infection groups, only a small amount of granulomas were seen in the liver, together with weak inflammatory response which showed a conversion toward fibrosis repair; however, in the high infection group, a small germinal layer structure was formed in the liver, surrounded by dense inflammatory cells. The absolute numbers of splenic NKT cells in the medium and high infection groups were (6.32 ± 0.53) × 10 ⁵ and (7.37 ± 1.72) × 10⁵, respectively, which were higher than those in the control group and the low infection group [(5.02 ± 1.08) × 10 ⁵ and (4.90 ± 1.27) × 10 ⁵, P < 0.01]. The proportions of splenic NKT1-type cells secreting IFN-γ in the medium and high infection groups were (13.93 ± 1.61)% and (10.9 ± 3.04)%, respectively, which were lower than those in the control group and the low infection group [(17.3 ± 3.18)% and (19.42 ± 2.82)%, P < 0.01)]; the proportions of splenic NKT2-type cells secreting IL-4 in the low, medium and high infection groups were (5.62 ± 0.58)%, (5.86 ± 1.43)% and (5.92 ± 0.94)%, respectively, which were higher than that in the control group [(4.37 ± 0.83) %, P < 0.05)]; the proportion of splenic NKT17-type cells secreting IL-17A in the low and medium infection groups were (7.02 ± 1.19)% and (6.62 ± 0.41)%, respectively, which were higher than those in the control group and the high infection group [(4.68 ± 0.73)% and (3.73 ± 1.04)%, P < 0.01]. The absolute numbers of splenic CD4 ⁺ NKT cells in the medium and high infection groups were (1.75 ± 0.36) × 10 ⁵ and (1.82 ± 0.24) × 10 ⁵, respectively, which were higher than those in the control group [(1.24 ± 0.26) × 10 ⁵, P < 0.01]; the proportions of splenic CD4 ⁺ NKT1-type cells secreting IFN-γ in the low, medium- and high infection groups were (19.42 ± 2.82)%, (14.40 ± 1.81)%, and (12.5 ± 2.96)%, respectively, which were lower than those in the control group [(21.16 ± 2.83) %,P < 0.01]; the proportions of splenic CD4 ⁺ NKT17-type cells secreting IL-17A in the low, medium and high infection groups were (18.73 ± 1.99)%, (16.06 ± 1.42)% and (14.41 ± 3.55)%, respectively, which were higher than that in the control group [(10.27 ± 1.79)%,P < 0.01]. The proportions of splenic CD8 ⁺ NKT cells secreting IFN-γ in the low, medium and high infection groups were (10.04 ± 0.92)%, (13.43 ± 0.92)% and (9.24 ± 1.37)%, respectively, which were lower than those in the control group [(15.83 ± 1.59)%,P < 0.01]; the proportions of splenic CD8 ⁺ NKT cells secreting IL-10 in the medium and high infection groups were (4.07 ± 0.48)% and (3.92 ± 0.75)%, respectively, which were higher than that in the control group [(2.85 ± 0.64) %, P < 0.05]. The proportions of splenic DN NKT cells secreting IFN-γ in the medium and high infection groups were (17.48 ± 2.50)% and (14.06 ± 3.95)%, respectively, which were lower than that in the control group [(25.18 ± 5.78)%, P < 0.01]. Conclusion After infection of different numbers of E. multilocularismetacestode, the mice spleen NKT cells and their subsets changed significantly. The mice in the low and middle infection groups showed the immune response predominately with the CD4⁺ NKT17 type to kill and clear the parasites, while mice in the high infection group responded largely with the mixed subsets of CD4⁺ NKT17, NKT2 and NKT10 type cells, and NKT cells showed an inhibitory phenotype, leading to the decline of NKT1-type immune response, which is in favour of the parasite surviving.