中、晚期妊娠胎盘单核-巨噬细胞经内皮穿越后免疫调节活性的比较

Changes of immunological activity of trans-endothelial trafficking monocyte-macrophages from the mid-term and late pregnancy placenta

目的 :观察中、晚期妊娠胎盘单核-巨噬细胞经内皮穿越分化后免疫调节活性的差异。方法:分离新生儿脐静脉内皮细胞,建立单层内皮细胞。分离中、晚期妊娠胎盘CD14+单核-巨噬细胞,与单层内皮细胞共培养诱导其分化。以ELISA法检测经内皮单层诱导分化前后单核-巨噬细胞培养上清中的IL-12、IL-10水平;以流式细胞术检测经内皮穿越而分化的细胞诱导同种异型脐血淋巴细胞CD69分子和核因子Foxp3的表达。结果:1晚期妊娠胎盘单核-巨噬细胞IL-12分泌水平显著高于、IL-10分泌水平显著低于中期妊娠胎盘单核-巨噬细胞,培养上清中IL-10/IL-12比值在晚期和中期妊娠胎盘单核-巨噬细胞中分别为2.59±0.71和71.4±8.49;经内皮单层穿越诱导分化后,中、晚期妊娠胎盘单核-巨噬细胞IL-12分泌水平均显著增高、IL-10分泌水平则显著降低,但晚期妊娠单核-巨噬细胞IL-12分泌水平提高倍数不及中期妊娠单核-巨噬细胞[(2.00±0.35)倍vs(8.91±1.49倍)],而IL-10分泌水平下降比中期妊娠显著[(92.1±8.2)%vs(57.8±4.98)%],导致IL-10/IL-12比值下降仅到0.11±0.04,而中期妊娠单核-巨噬细胞依然保持IL-10/IL-12比值>1(3.16±0.96);2两类细胞经内皮(逆)穿越后都能激活同种异型脐血T淋巴细胞表达CD69,其中晚期妊娠胎盘来源细胞的激活作用显著高于中期妊娠胎盘来源的细胞[(73.59±3.52)%vs(45.32±7.47)%];3在与同种异型脐血淋巴细胞共育的过程中,与晚期妊娠胎盘来源的细胞相比,中期妊娠胎盘来源的细胞则能诱导T细胞分化形成更多的Foxp3+T细胞。结论 :中、晚期妊娠胎盘单核-巨噬细胞经内皮单层穿越系统的诱导培养,可分化成具有不同免疫学特性的细胞。晚期妊娠胎盘来源的细胞经诱导分化后能刺激T细胞充分活化,表达高水平CD69,而中期妊娠胎盘来源的细胞经分化后具有显著地诱导Treg的免疫调节活性。

Objective：To observe the differences of immunological activity of trans-endothelial trafficking monocyte-macrophage from mid-term placenta and late placenta. Methods：Umbilical vein endothelial cells were isolated and cultured for establishing endothelial cell monolayer. CD14＋monocyte-macrophage cells were separated from the mid-term placenta and late placenta and induced to differentiate by trafficking through the endothelial cell monolayer. We detected IL-12 and IL-10 levels of the culture supernatants with ELISA method before and after trafficking of monocyte-macrophages through endothelial cell monolayer. Flow cytometry was performed to detect the expressions of CD69 molecule and nuclear factor Foxp3 by allogeneics cord blood T lymphocyte co-cultured with differentiated monocyte-macrophages through transendothelial trafficking. Results： ① In the culture supernatant of monocyte-macrophages from late pregnancy placenta,IL-12 level was remarkably higher,while IL-10 level remarkably lower than those in the supernatant of the cells from mid-term pregnancy placenta,respectively. The ratio of IL-10 / IL-12 was 2.59 ±0.71,remarkably lower than that in the supernatant of cells from mid-term placenta（71.4 ± 8.49）. After trafficking through endothelial monolayer,IL-12 level increased,while IL-10 level decreased significantly in the supernatants of both cells from lateplacenta and mid-term placenta,and IL-10 level decreased more significantly in the supernatants of the cells from late pregnancy placenta than that from mid-term pregnancy placenta [（92.1 ± 8.2）% vs.（57.8 ± 4.98）%], so that IL-10 / IL-12 ratio dropped down to less than 1（0.11 ± 0.04） while the ratio remained more than 1（3.16 ± 0.96） in the supernatants of the cells from mid-term placenta. ②After trafficking through endothelial monolayer,both cells from late placenta and mid-term pregnancy placenta activated allogenic cord blood T lymphocytes to express CD69,and CD69 expression level of T cells stimulated by the cells from late pregnancy placenta was significantly higher than that by the cells from mid-term placenta [（73.59 ± 3.52）% vs（45.32 ± 7.47）%]. ③After trafficking through endothelial monolayer,compared with the cells from late placenta,the cells from mid-term placenta induced allogenic cord blood T cells to differentiate into more Foxp3＋T cells. Conclusion：By trafficking through endothelial monolayer,monocyte-macrophages from mid-term and late pregnancy placenta differentiate into different cells with distinctively immunologic characteristics. After trafficking through endothelial monolayer,monocyte-macrophages from late pregnancy placenta differentiate into a kind of immunologically stimulatory cells which could fully stimulate T cells to activate and express high level of CD69,while monocyte-macrophages from mid-term pregnancy placenta differentiate into a kind of immunologically regulatory cells which are capable for inducing Treg.