目的 探讨孕期刚地弓形虫感染对滋养层细胞及蜕膜免疫细胞表面CD73表达的影响，并分析其与不良妊娠结局的关系。方法 收集健康早孕期正常流产的蜕膜和绒毛组织4个，采用聚蔗糖-泛影葡胺淋巴细胞分离液分离蜕膜单个核细胞，percoll法分离滋养层细胞，将纯化的蜕膜单个核细胞和滋养层细胞分别分为对照组和感染组，每组1 × 10⁷个，感染组按细胞与弓形虫3∶1感染，感染后24 h，采用流式细胞术检测滋养层细胞、蜕膜自然杀伤细胞（decidual natural killer，dNK)、蜕膜巨噬细胞（decidual macrophage，dMφ）和蜕膜树突状细胞（decidual dendritic cell，dDC）表面CD73表达。将分离的滋养层细胞分为对照组和感染组，每组1 × 10⁷个，感染组按细胞与弓形虫1∶1感染，感染后24 h提取蛋白，蛋白质免疫印记（Western blotting）分析弓形虫感染后滋养层细胞CD73蛋白的相对表达水平。野生型C57BL/6J雌鼠60只与雄鼠30只，CD73基因敲除（CD73^-/-）雌鼠60只与雄鼠30只均分别按雌雄比为2∶1随机合笼过夜。孕鼠随机均分为野生型对照组和野生型感染组，CD73^-/-对照组和CD73^-/-感染组，孕8 d，感染组孕鼠分别经腹腔注射400个弓形虫速殖子，对照组注射等量生理盐水。孕14 d，解剖子宫并观察胎盘、胎鼠情况，计算异常胎率。免疫组化染色检测胎盘组织中CD73的表达。采用GraphPad Prism 9.0软件进行统计学分析，组间数据分析采用独立样本Student’s t检验。结果 流式细胞术结果显示，对照组滋养层细胞表面CD73阳性细胞比例为（39.25 ± 1.43）%，高于感染组的（31.27 ± 2.79）%（t = 5.31，P < 0.05），对照组dDC、dMφ和dNK细胞表面CD73阳性细胞比例分别为（38.17 ± 7.71）%、（25.80 ± 6.85）%、（9.39 ± 0.75）%，高于感染组的（36.33 ± 7.64）%、（20.58 ± 5.18）%、（7.74 ± 1.08）%（t = 4.25、5.24、3.57，均P < 0.05）；Western blotting结果显示，对照组滋养层细胞CD73蛋白相对表达水平为0.79 ± 0.07，较感染组的0.38 ± 0.05显著下调（t = 24.80，P < 0.01）；免疫组化结果显示，野生型对照组胎盘组织阳性细胞呈棕色集中表达在胎盘侧，尤其近血管处，野生型感染组阳性细胞较对照组少。野生型感染组小鼠胎盘组织CD73表达灰度值为9.79 ± 0.30，较野生型对照组的20.20 ± 4.60显著降低（t = 3.89，P < 0.05）。野生型对照组和CD73^-/-对照组胎盘胎鼠发育良好，有光泽，子宫圆润饱满，野生型感染组和CD73^-/-感染组孕鼠明显缺血，伴有严重淤血，胎鼠与胎盘不成形，出现吸收胎鼠现象，胎盘发育不良。野生型感染组小鼠胎盘和胎鼠体质量分别为（0.05 ± 0.01）g和（0.07 ± 0.03）g，低于野生型对照组的（0.07 ± 0.01）g和（0.13 ± 0.08）g（t = 3.22、5.36，均P < 0.01）；CD73^-/-对照组分别为（0.07 ± 0.04）g 和（0.12 ± 0.01）g，与野生型对照组差异无统计学意义（t = 0.62、0.13，均P > 0.05）；CD73^-/-感染组分别为（0.03 ± 0.00）g 和（0.01 ± 0.00）g，较野生型感染组显著减轻（t = 3.10、5.08，均P < 0.01）。野生型对照组和CD73^-/-对照组异常胎率均为0，野生型感染组为（55.73 ± 0.18）%，较野生型对照组显著增加（t = 7.79，P < 0.01），CD73^-/-感染组为（76.00 ± 0.20）%，较野生型感染组显著增加（t = 2.44，P < 0.05）。结论 孕早期刚地弓形虫感染可显著下调母胎微环境中滋养层细胞及蜕膜免疫细胞（dNK、dMφ和dDC）表面CD73的表达，且CD73表达下调与弓形虫感染致不良妊娠的发生密切相关。

Objective To investigate the effect of Toxoplasma gondii infection during pregnancy on CD73 expression in trophoblasts and decidual immune cells and to examine the association of CD73 expression with adverse pregnancy. Methods Four healthy decidua and villi tissue specimens in normal abortions at early pregnancy were collected. Decidual mononuclear cells were isolated with Ficoll-Paque lymphocyte separation solutions, and trophoblasts were isolated with the Percoll method. Purified decidual mononuclear cells and trophoblasts were divided into the control and infection groups, with 1 × 10⁷ cells in each group, and cells in the infection group were infected with T. gondii at a ratio of 3∶1 for 24 hours. Then, the CD73 expression was detected on the surface of trophoblasts, decidual natural killer (dNK) cells, decidual macrophage (dMφ) and decidual dendritic cell (dDC) using flow cytometry. Trophoblasts were divided into the control and infection groups, with 1 × 10⁷ cells in each group, and cells in the infection group were infected with T. gondii at a ratio of 1∶1 for 24 hours. Then, total protein was extracted from cells, and the relative CD73 protein expression was determined in trophoblasts using Western blotting post-infection with T. gondii. Sixty wild-type female mice of the C57BL/6J strain and 30 male mice of the C57BL/6J strain, and 60 female CD73^-/- mice and 30 male CD73^-/- mice were randomly caged overnight at a female to male ratio of 2∶1, respectively. Wild-type pregnant mice were randomly divided into the Wild-type control and Wild-type infection groups, and CD73^-/- pregnant mice were randomly divided into the CD73^-/- control and infection groups. Pregnant mice in the two infection groups were intraperitoneally injected with 400 T. gondii tachyzoites on day 8 of pregnancy, and pregnant mice in the two control groups were injected with the same volume of physiological saline. Mouse uterus was dissected on day 14 of pregnancy, and the placentas and fetal mice were observed, with the percentage of fetal abnormality estimated. The CD73 expression was detected in placental tissues using immunohistochemical staining. All statistical analyses were performed using the software GraphPad Prism 9.0, and differences of means were tested for statistical significance with Student’s t test for independent samples. Results Flow cytometry detected that the proportion of CD73-positive trophoblasts in total trophoblasts was (39.25 ± 1.43)% in the control group and was higher than that in the infection group (31.27 ± 2.79)% in the infection group (t = 5.31, P < 0.05). The proportions of CD73-positive cells were (38.17 ± 7.71)%, (25.80 ± 6.85)% and (9.39 ± 0.75)% on the surface of dDCs, dMφ and dNK cells in the control group higher, all were higher than that in the infection group (36.33 ± 7.64)%, (20.58 ± 5.18)% and (7.74 ± 1.08)% in the infection group (t = 4.25, 5.24 and 3.57; P < 0.05). Western blotting determined that the relative CD73 protein expression was 0.79 ± 0.07 in trophoblasts in the control group and 0.38 ± 0.05 in the infection group (t = 24.80, P < 0.01), and immunohistochemical staining showed that wild-type control placental tissue-positive cells were brown in color and expressed centrally on the lateral side of the placenta, especially near blood vessels, wild-type infected group had fewer positive cells were fewer in the wild-type infected group than in the control group, the grayscale value of CD73 expression was 9.79 ± 0.30 in mouse placental tissues in the wild-type infection group and 20.20 ± 4.60 in the wild-type control group (t = 3.89, P < 0.05). The placentas and fetuses of wild-type control and CD73^-/- control groups were well-developed, shiny, and the uterus was round and full, whereas pregnant rats of wild-type infected and CD73^-/- infected groups were obviously ischemic with severe bruising, and the fetuses and placentas were not shaped, and the fetuses were absorbed, and the placentas were underdeveloped. The mouse placenta and fetal body weights were (0.05 ± 0.01) g and (0.07 ± 0.03) g in the wild-type infection group, and (0.07 ± 0.01) g and (0.13 ± 0.08) g in the wild-type control group (t = 3.22 and 5.36, both P < 0.01). The mouse placenta and fetal body weights were (0.07 ± 0.04) g and (0.12 ± 0.01) g in the CD73^-/- control group, which were comparable to those in the wild-type control group (t = 0.62 and 0.13, both P > 0.05), and were (0.03 ± 0.00) g and (0.01 ± 0.00) g in the CD73^-/- infection group, which significantly reduced as compared with those in the wild-type infection group (t = 3.10 and 5.08, both P < 0.01). The proportions of fetal abnormality were 0 in the wild-type control group and CD73^-/- group, (55.73 ± 0.18)% in the wild-type infection group, which was significantly higher than in the wild-type control group (t = 7.79, P < 0.01), and was (76.00 ± 0.20)% in the CD73^-/- infection group, which significantly increased as compared with that in the wild-type infection group (t = 2.44, P < 0.05). Conclusion T. gondii infection at early pregnancy may remarkably down-regulate CD73 expression on the surface of trophoblasts and decidual immune cells (dNK cells, dMφ and dDC) in the maternal-fetal microenvironment, and CD73 down-regulation is strongly associated with adverse pregnancy caused by T. gondii infection.