重度子痫前期患者血清外泌体对人正常蜕膜免疫细胞功能的影响

doi:10.12280/gjfckx.20241045

摘要/Abstract

摘要：

目的： 探讨重度子痫前期（preeclampsia，PE）患者血清外泌体对人正常蜕膜组织免疫细胞功能的影响。方法： 收集同期住院分娩的重度PE患者（PE组）及正常孕妇（NC组）血清标本各5例，并提取血清中外泌体；收集行人工流产术患者的蜕膜组织共6例，提取并纯化人正常早孕流产蜕膜组织中蜕膜自然杀伤（decidual natural killer，dNK）细胞、巨噬细胞（macrophages，M?）、CD4+T细胞和CD8+T细胞。蛋白质印迹（Western blotting）检测外泌体中胎盘源性特异性标志蛋白胎盘碱性磷酸酶（placental alkaline phosphatase，PLAP）表达；检测摄取血清外泌体后蜕膜中主要免疫细胞（dNK、M?、CD4+T、CD8+T）的相关功能。结果： 2组血清外泌体形态典型，且均表达外泌体特征性蛋白及PLAP。2组dNK、M?、CD4+T、CD8+T细胞均能正常摄取血清外泌体。与NC组相比，PE组摄取血清外泌体后，dNK分泌的白细胞介素-8（interleukin-8，IL-8）、IL-10、干扰素诱导蛋白-10（interferon-inducible protein-10，IP-10）、转化生长因子-β（transforming growth factor-β，TGF-β）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor，GM-CSF）、白血病抑制因子（leukemia inhibitory factor，LIF）水平均降低；M1型M?占比增加，M?分泌IL-12、IL-23水平升高，IL-4、IL-10水平降低；CD4+T、CD8+T细胞增殖率降低、凋亡率升高，调节性T细胞（regulatory T cell，Treg）占比下降，分泌的肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、γ干扰素（interferon-γ，IFN-γ）水平升高，IL-4、IL-10水平降低，且细胞中程序性死亡受体1（programmed death-1，PD-1）、T细胞免疫球蛋白和含黏蛋白结构域蛋白3（T-cell immunoglobulin and mucin domain-containing protein 3，Tim-3）水平降低，差异均有统计学意义（均P<0.05）。结论： 重度PE患者胎盘源性血清外泌体能够影响母胎界面多种蜕膜免疫细胞相关功能发生改变。

关键词: 先兆子痫, 外泌体, 蜕膜, 免疫, 细胞, 胎盘, 功能状态

Abstract:

Objective: To investigate the effect of serum exosomes from patients with severe preeclampsia (PE) on the immune cell function of normal decidual tissue. Methods: Serum samples were collected from 5 severe PE patients (PE group) and 5 normal pregnant women (NC group) who were hospitalized for delivery during the same period, and serum exosomes were extracted. Six cases of decidual tissue from patients undergoing induced abortion were collected, and dNK cells, M?, CD4+T cells, and CD8+T cells were extracted and purified from the decidual tissue of normal early pregnancy miscarriage. Western blotting was used to detect the expression of PLAP, a placental specific marker protein in the serum exosomes, and detect the relevant functions of major immune cells (dNK, M?, CD4+T, CD8+T) in the decidua after uptake of serum exosomes. Results: The morphology of two groups of serum exosomes was typical, and both expressed characteristic proteins of exosomes and PLAP. Both groups of dNK, M?, CD4+T and CD8+T cells were able to uptake serum exosomes normally. Compared with the NC group, the PE group showed a decrease in the levels of IL-8, IL-10, IP-10, TGF-β, VEGF, GM-CSF, and LIF secreted by dNK after ingesting serum exosomes. The proportion of M1 type M? increased, and the secretion of IL-12 and IL-23 levels by M? increased, while the levels of IL-4 and IL-10 decreased. The proliferation rate of CD4+T and CD8+T cells decreased, the apoptosis rate increased, the proportion of Treg cells decreased, the levels of TNF-α and IFN-γ secreted increased, the levels of IL-4 and IL-10 decreased, and the levels of PD-1 and Tim-3 in cells decreased, with statistically significant differences (all P<0.05). Conclusions: Placental derived serum exosomes from severe PE patients can affect various decidual immune cell related functions at the maternal fetal interface.

Key words: Pre-eclampsia, Exosomes, Decidua, Immunity, Cells, Placenta, Functional status

杨洋, 马媛, 陈宥艺, 赵静, 马文娟. 重度子痫前期患者血清外泌体对人正常蜕膜免疫细胞功能的影响[J]. 国际妇产科学杂志, 2025, 52(2): 143-152.

YANG Yang, MA Yuan, CHEN You-yi, ZHAO Jing, MA Wen-juan. The Effect of Serum Exosomes from Patients with Severe Preeclampsia on the Function of Normal Decidual Immune Cells in Humans[J]. Journal of International Obstetrics and Gynecology, 2025, 52(2): 143-152.

图/表 6

图1 血清中外泌体提取、鉴定及胎盘源性检测注：A 透射电镜下可见外泌体，标尺=200 nm；B 外泌体粒径分布[纵坐标：原始颗粒浓度（单位：107个颗粒/mL；每刻度=0.5 mL）；横坐标：粒径大小（单位：nm；每刻度=100 nm）]；C Western blotting检测外泌体标记蛋白CD63和CD9、阴性标记蛋白β-actin、Cyt-c以及胎盘源性特异性蛋白PLAP的表达。

图2 流式细胞术分选人蜕膜dNK、M?、CD4+T、CD8+T细胞纯度注：A CD56+CD16-dNK细胞纯度[横坐标：CD56荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：CD16荧光信号相对强度值（每刻度=以10为底的单倍递增指数）]；B M?纯度[横坐标：F4/80荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：粒子数（每刻度=20个）/侧向散射光测量值（每刻度=10×104）]；C CD4+T细胞纯度[横坐标：CD4荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：粒子数（每刻度=40个）/侧向散射光测量值（每刻度=100×103）]；D CD8+T细胞纯度[横坐标：CD8荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：粒子数（每刻度=40个）/侧向散射光测量值（每刻度=10×104）]。

图3 dNK细胞对血清外泌体的摄取及分泌功能情况注：A dNK细胞摄取外泌体后的荧光显微镜图（×400）；B 2组dNK细胞摄取血清外泌体后分泌细胞因子水平比较，* 与NC组比较，P<0.05。

图4 M?对血清外泌体的摄取、极化及分泌功能情况注：A M?摄取外泌体后的荧光显微镜图（×400）；B 流式细胞术检测2组M?摄取外泌体后M1/M2型M?占比[横坐标：CD86荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：CD206荧光信号相对强度值（每刻度=以10为底的单倍递增指数）]；C 2组M1/M2型M?占比比较；D 2组M?摄取血清外泌体后分泌细胞因子水平比较。* 与NC组比较，P<0.05。

图5 CD4+T细胞对血清外泌体摄取、细胞功能及免疫检查点分子蛋白表达注：A CD4+T细胞摄取外泌体后的荧光显微镜图（×400）；B 流式细胞术检测2组CD4+T细胞摄取外泌体后增殖率[横坐标：FITC-A荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：粒子数（每刻度=20个）]；C 流式细胞术检测2组CD4+T细胞摄取外泌体后凋亡率[横坐标：FITC-A荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：碘化丙啶标记荧光信号相对强度值（每刻度=以10为底的单倍递增指数）]；D 流式细胞术检测CD4+T细胞摄取外泌体后CD4+CD25+FOXP3+Treg占比情况[横坐标：CD25荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：FOXP3荧光信号相对强度值（每刻度=以10为底的单倍递增指数）]；E 2组CD4+T细胞摄取血清外泌体后分泌细胞因子水平比较；F 2组CD4+T细胞摄取血清外泌体后细胞中免疫检查点分子蛋白水平比较。* 与NC组比较，P<0.05。

图6 CD8+T细胞对血清外泌体摄取、细胞功能及免疫检查点分子蛋白表达注：A CD8+T细胞摄取外泌体后的荧光显微镜图（×400）；B 流式细胞术检测2组CD8+T细胞摄取外泌体后增殖率[横坐标：FITC-A荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：粒子数（每刻度=20个）]；C 流式细胞术检测2组CD8+T细胞摄取外泌体后凋亡率[横坐标：FITC-A荧光信号相对强度值（每刻度=以10为底的单倍递增指数）；纵坐标：碘化丙啶标记荧光信号相对强度值（每刻度=以10为底的单倍递增指数）]；D 2组CD8+T细胞摄取血清外泌体后分泌细胞因子水平比较；E 2组CD8+T细胞摄取血清外泌体后细胞中免疫检查点分子蛋白水平比较。* 与NC组比较，P<0.05。

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