Tim-3、PD-1调节外周血单核细胞功能参与URSA发病机制探讨
发布时间：2018-05-25 06:54
本文选题：Tim-3 + PD-1 ； 参考：《山西医科大学》2017年硕士论文
【摘要】：目的:1.探讨URSA患者外周血单核细胞上Tim-3、PD-1的表达和血浆中IL-4、IL-6、IL-10、IL-12、IFN-γ的含量,为研究URSA发病机制提供理论依据。2.探讨Tim-3、PD-1对单核细胞功能的调节,初步探讨Tim-3、PD-1在URSA中可能的作用机制。3.Tim-3或PD-1有望成为URSA免疫治疗的新靶点。方法:选取30例健康早孕妇女作为正常早孕组(NP组),30例URSA早孕患者为URSA组;采用Ficoll密度梯度离心法从研究对象的外周血中分离单个核细胞(peripheral blood mononuclear cell,PBMC),流式细胞分析技术(Flow cytometry,FCM)检测2组孕妇外周血中CD14~+单核细胞(monocyte,MC)上Tim-3、PD-1的表达水平;酶联免疫吸附法(Enzyme linked immunosorbent assay,ELISA)检测外周血浆中IL-4、IL-6、IL-10、IL-12、IFN-γ的含量;从两组中各选取10例通过免疫磁珠技术阳性分选CD14~+单核细胞,然后分别加入anti-Tim-3、anti-PD-1或anti Tim-3+anti-PD-1阻断后,给予LPS/R848(5ug/m L)刺激48小时后流式细胞技术检测CD14~+单核细胞上PD-1或Tim-3表达水平及细胞内IL-12分泌情况,采用Graph Pad Prism5.0统计软件处理相关数据。结果:1.与NP组相比,URSA组患者外周血CD14~+MC上Tim-3、PD-1表达降低(P0.05);相关性分析Tim-3和PD-1呈正相关(P0.01)。2.ELISA检测结果分析:URSA组外周血浆中IL-4、IL-10浓度较NP组明显降低(P0.01);IFN-γ、IL-6水平升高(P0.01);MC相关细胞因子IL-12在URSA组表达水平增高,IL-12与Th2型细胞因子(IL-4、IL-10)呈负相关(r=-0.6714,r=-0.5593,P0.01);IL-12与Th1型细胞因子(IL-6、IFN-γ)呈正相关(r=0.7740,r=0.5590,P0.01)。3.Tim-3和PD-1与细胞因子相关性分析:Tim-3和PD-1与IL-4、IL-10呈正相关(r=0.6343/0.426,r=0.5356/0.4529,P0.05);与IL-6、IFN-γ呈负相关(r=-0.5153/-0.5649,P0.05,r=-0.4518/-0.6543,P0.01),与IL-12呈负相关(r=-0.5216,r=-0.540,P0.01)。4.阻断Tim-3通路后,NP组和URSA组外周血CD14~+MC上PD-1表达水平降低;URSA组CD14~+MC上PD-1表达水平低于正常早孕组;同样,体外阻断PD-1信号后,两组CD14~+MC上Tim-3表达含量降低,URSA组低于正常早孕组(P0.01)。5.与正常早孕相比,URSA组外周血CD14~+MC内IL-12含量增加;阻断Tim-3或PD-1后,两组患者外周血CD14~+MC内IL-12分泌量增加;在正常早孕组中,阻断Tim-3较阻断PD-1后MC内IL-12表达量增加,而在URSA组中,单一阻断Tim-3或PD-1,CD14~+MC内IL-12分泌水平变化无明显差异(P=0.0515),两组同时阻断Tim-3和PD-1后,CD14~+MC内IL-12含量较单独阻断Tim-3或PD-1时增高明显(P0.01)。结论:1.Tim-3和PD-1参与正常妊娠的维持,CD14~+MC上Tim-3和PD-1表达失衡可能与URSA的发生有关。2.在URSA患者外周血中Th1/Th2平衡向Th1偏移,Tim-3和PD-1可能通过抑制MC对IL-12的分泌,间接调节相关细胞因子的表达,介导Th1/Th2平衡向Th1漂移,从而参与URSA的发生。3.通过体外阻断Tim-3和PD-1信号,发现Tim-3和PD-1抑制MC分泌IL-12,从而负性调节MC功能,间接调控Th1/Th2平衡。
[Abstract]:Purpose 1. To investigate the expression of Tim-3 PD-1 on peripheral blood monocytes of URSA patients and the level of IL-12 IFN- 纬 in plasma of IL-4, IL-6, IL-10 and IL-10, and to provide a theoretical basis for studying the pathogenesis of URSA. To investigate the regulation of Tim-3P PD-1 on monocyte function, and to explore the possible mechanism of Tim-3P PD-1 in URSA. 3. Tim-3 or PD-1 may be a new target of URSA immunotherapy. Methods: 30 healthy early pregnant women were selected as normal early pregnancy group and 30 patients with URSA early pregnancy as URSA group. Ficoll density gradient centrifugation was used to isolate mononuclear blood mononuclear cells from peripheral blood of the study subjects. Flow cytometric analysis (FCM) was used to detect the expression of Tim-3G PD-1 on the peripheral blood of the two groups of pregnant women. Enzyme linked immunosorbent assay (enzyme linked immunosorbent assay) was used to detect the level of IL-4, IL-6, IL-10, IL-12, IFN- 纬 in peripheral plasma, 10 cases of CD14 ~ monocytes were selected from each group by immunomagnetic bead technique, and then anti-Tim-3 anti-PD-1 or anti Tim-3 anti-PD-1 were added respectively. Flow cytometry was used to detect the expression of PD-1 or Tim-3 and intracellular IL-12 secretion in CD14- monocytes after 48 hours of stimulation with LPS/R848(5ug/m L. The relevant data were processed by Graph Pad Prism5.0 software. The result is 1: 1. Compared with NP group, the expression of Tim-3P-1 on peripheral blood CD14 ~ MC of USA group decreased P0.05.The correlation analysis showed that there was a positive correlation between Tim-3 and PD-1. The results of Elisa analysis of IL-4 IL-10 in peripheral plasma of the control group were significantly lower than that of NP group. The level of IFN- 纬 IL-6 in the peripheral blood of the control group was significantly lower than that in the NP group. The level of IL-3PD-1 in the peripheral blood was significantly lower than that in the NP group, and the increase of the level of IL-4- 纬 IL-6 in the plasma of the control group was significantly lower than that in the NP group. 鍦║RSA缁勮〃杈炬按骞冲楂，
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