目的 探索孕期刚地弓形虫感染对蜕膜免疫细胞T细胞免疫球蛋白黏蛋白-3（Tim-3）表达的影响及差异关系。方法 取孕鼠分为野生型（WT）孕鼠对照组、WT孕鼠感染组和Tim-3^-/-孕鼠感染组，每组20只。2个感染组小鼠于孕8 d，经腹腔感染250条弓形虫RH株速殖子（250 μl/鼠），对照组注射等量生理盐水。于孕14天，观察各组孕鼠精神状态；安乐死处死各组孕鼠后，解剖并记录妊娠结局，计算异常胎鼠率；取孕鼠胎盘、胎鼠并称重，比较3组间不良妊娠结局差异。取早孕期自愿终止妊娠的健康孕妇的蜕膜组织，采用密度梯度离心法收集蜕膜单个核细胞，分为对照组和感染组（每组1 × 10⁷个细胞）。感染组按细胞∶弓形虫为3∶1的比例进行感染，对照组加入等量的不含弓形虫的培养基，20 h后收集两组细胞，采用流式细胞术检测蜕膜自然杀伤（dNK）细胞、蜕膜巨噬细胞（dMϕ）、蜕膜髓源性抑制细胞（dMDSC）和蜕膜树突状细胞（dDC）Tim-3的表达水平。采用免疫磁珠自蜕膜单个核细胞中分别分选出dNK细胞、dMϕ、dMDSC和dDC，将各类细胞分为感染组和对照组。感染组按细胞∶弓形虫为3∶1的比例进行感染，对照组加入等量培养基，20 h后收集细胞，提纯蛋白，利用蛋白质免疫印迹（Western blotting）分析各类蜕膜免疫细胞Tim-3的表达水平。组间差异比较采用独立样本t检验（体内实验）或配对t检验（体外实验）。结果 WT感染组孕鼠胎盘和胎鼠质量分别为（83.13 ± 9.13）和（96.81 ± 11.63）mg，较WT对照组的（105.18 ± 9.64）和（122.86 ± 3.67）mg明显下降（t = 4.394、5.655，P < 0.01)；Tim-3^-/-感染组孕鼠的胎盘和胎鼠质量分别为（66.31 ± 9.00）和（74.32 ± 6.33）mg，均低于WT感染组（t = 3.471，P < 0.01；t = 4.495，P < 0.05）。WT感染组流产率为（30.14 ± 9.87）%，较WT对照组（无流产胎鼠）明显升高（t = 8.076，P < 0.01)；Tim-3^-/-感染组流产率为（66.85 ± 26.17）%，高于WT感染组（t = 3.473，P < 0.01）。Tim-3^-/-感染组孕鼠的不良妊娠结局较WT感染组孕鼠明显加重。流式细胞术检测结果显示，感染组人dNK细胞、dMϕ、dMDSC、dDC中的Tim-3阳性细胞占比分别为（12.22 ± 4.61）%、（23.87 ± 5.26）%、（10.07 ± 1.02）%、11.01 ± 4.10）%，均低于对照组的（24.50 ± 7.10）%、（41.23 ± 3.84）%、（17.53 ± 0.65）%、（18.53 ± 4.93）%（t = 7.717、5.865、7.798、9.592，均P < 0.05）。Western blotting检测结果显示，感染组人dNK细胞、dMϕ、dMDSC、dDC中的Tim-3相对表达水平分别为0.69 ± 0.09、0.74 ± 0.09、0.79 ± 0.08、0.84 ± 0.04，均低于对照组的1.03 ± 0.06、0.98 ± 0.06、0.95 ± 0.02、0.97 ± 0.06（t = 7.562、4.099、4.398、5.455，P < 0.05或P < 0.01）。流式细胞术检测结果比较显示，弓形虫感染后，Tim-3在dNK细胞内的降幅最大，为（50.82 ± 5.92）%；在dMDSC和dMϕ内的降幅分别为（42.37 ± 7.96）%、（42.08 ± 12.31）%；在dDC内的降幅最小，为（41.45 ± 7.64）%。Western blotting检测比较结果与流式细胞术基本一致，即弓形虫感染后，各蜕膜免疫细胞Tim-3表达均下调，在dNK细胞内的降幅最大，为（33.06 ± 8.52）%；在dMϕ内次之，为（22.84 ± 10.64）%；在dMDSC和dDC内的降幅较小，分别为（16.25 ± 7.44）%和（13.6 ± 4.42）%。结论 早孕期刚地弓形虫感染可显著下调母胎界面各类主要蜕膜免疫细胞Tim-3的表达水平，其中对dNK细胞影响最大，其他依次为dMϕ、dMDSC、dDC。弓形虫感染后各类蜕膜免疫细胞Tim-3表达水平的下调在不良妊娠结局的发生中发挥重要作用。

Objective To investigate the effect of Toxoplasma gondii infection during pregnancy on the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in decidual immune cells and differential relationships. Methods Pregnant mice were assigned into the wild-type (WT) pregnant control group, the WT pregnant infection group, and the Tim-3^-/- pregnant infection group, of 20 mice each group. Mice in two infection groups were intraperitoneally inoculated with 250 tachyzoites of the T. gondii RH strain suspended in 250 μl of diluents at gestational ages of 8 days, while mice in the control group received an equal volume of physiological saline. The mental state of pregnant mice was observed in each group at gestational ages of 14 days, and all pregnant mice were sacrificed with euthanasia, dissected, recorded for pregnancy outcomes and calculate for the proportion of abnormal fetuses. Placentas and fetuses were harvested from pregnant mice and weighed, and the differences in adverse pregnancy outcomes were compared among three groups. Decidual tissues were collected from healthy pregnant women undergoing voluntary termination of pregnancy in the first trimester, and decidual mononuclear cells were isolated using density gradient centrifugation and divided into a control group and an infection group, with 1 × 10⁷ cells per group. Cells in the infection group were challenged with T. gondii at a cell-to-parasite ratio of 3 ∶ 1, whereas cells in the control group were treated with an equal volume of T. gondii-free culture media. Cells in two groups were harvested 20 hours post-incubation, and the expression of Tim-3 was detected on decidual natural killer (dNK) cells, decidual macrophages (dMϕ), decidual myeloid-derived suppressor cells (dMDSCs), and decidual dendritic cells (dDCs) using flow cytometry. dNK cells, dMϕ, dMDSCs and dDCs were further sorted from decidual mononuclear cells with immunomagnetic beads, and each cell type was subdivided into an infection subgroup and a control subgroup. Cells in the infected subgroups were infected with T. gondii at a cell-to-parasite ratio of 3∶1, and those in control subgroups were incubated with an equal volume of parasite-free culture media. Cells were harvested 20 hours post-incubation for protein purification, and Tim-3 expression in each type of decidual immune cells was determined using Western blotting assay. Between-group differences were tested for statistical significance with independent-sample t-test for in vivo experiments or paired-sample t-test for in vitro experiments. Results The placental [(83.13 ± 9.13) mg vs. (105.18 ± 9.64) mg; t = 4.394, P < 0.01] and fetal weights [(96.81 ± 11.63) mg vs. (122.86 ± 3.67) mg; t = 5.655, P < 0.01] were significantly lower in the WT infection group than in the WT control group, and the placental [(66.31 ± 9.00) mg vs. (83.13 ± 9.13) mg; t = 3.471, P < 0.01] and fetal weights [(74.32 ± 6.33) mg vs. (96.81 ± 11.63) mg; t = 4.495, P < 0.05] were significantly lower in the Tim-3^-/- infection group than in the WT infection group. No aborted fetuses were observed in the WT control group, and the abortion rate was significantly higher in the WT infection group [(30.14 ± 9.87)%] than that in the WT control group (t = 8.076, P < 0.01), while the abortion rate was higher in the Tim-3^-/- infection group [(66.85 ± 26.17)%] than in the WT infection group (t = 3.473, P < 0.01). Adverse pregnancy outcomes were significantly aggravated in pregnant mice in the Tim-3^-/- infection group than in the WT infection group. Flow cytometry measured lower percentages of Tim-3 positive cells in human dNK cells [(12.22 ± 4.61)% vs. (24.50 ± 7.10)%; t = 7.717, P < 0.05], dMϕ [(23.87 ± 5.26)% vs. (41.23 ± 3.84)%; t = 5.865, P < 0.05], dMDSCs [(10.07 ± 1.02)% vs. (17.53 ± 0.65)%; t = 7.798, P < 0.05] and dDCs [(11.01 ± 4.10)% vs. (18.53 ± 4.93)%; t = 9.592, P < 0.05] in the infection group than in the control group, and Western blotting assay determined lower relative Tim-3 expression in human dNK cells [(0.69 ± 0.09) vs. (1.03 ± 0.06); t = 7.562, P < 0.01], dMϕ [(0.74 ± 0.09) vs. (0.98 ± 0.06); t = 4.099, P < 0.05], dMDSCs [(0.79 ± 0.08) vs. (0.95 ± 0.02); t = 4.398, P < 0.05], and dDCs [(0.84 ± 0.04) vs. (0.97 ± 0.06); t = 5.455, P < 0.05] in the infection group than in the control group, and flow cytometry measured the largest reduction [(50.82 ± 5.92)%] in Tim-3 expression in dNK cells following T. gondii infections, with (42.37 ± 7.96)% and (42.08 ± 12.31)% reductions in dMDSCs and dMϕ, and the smallest decrease in dDC cells [(41.45 ± 7.64)%]. Like flow cytometric analysis, Western blotting assay determined a reduction in Tim-3 expression in all types of decidual immune cells following T. gondii infections, with the largest reduction in dNK cells [(33.06 ± 8.52)%], followed by in dMϕ [(22.84 ± 10.64)%], and mild reductions in dMDSCs [(16.25 ± 7.44)%] and dDCs [(13.6 ± 4.42)%]. Conclusion T. gondii infections in the first trimester significantly downregulates Tim-3 expression in major decidual immune cell subsets at the maternal-fetal interface, with the most pronounced effect on dNK cells, followed by dMϕ, dMDSCs, and dDCs. Downregulation of Tim-3 expression in various types of decidual immune cells following T. gondii infection may play an important role in the development of adverse pregnancy outcomes.