本文選題：Tim-3 + PD-1��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:1.探討URSA患者外周血單核細(xì)胞上Tim-3、PD-1的表達(dá)和血漿中IL-4、IL-6、IL-10、IL-12、IFN-γ的含量,為研究URSA發(fā)病機(jī)制提供理論依據(jù)。2.探討Tim-3、PD-1對(duì)單核細(xì)胞功能的調(diào)節(jié),初步探討Tim-3、PD-1在URSA中可能的作用機(jī)制。3.Tim-3或PD-1有望成為URSA免疫治療的新靶點(diǎn)。方法:選取30例健康早孕婦女作為正常早孕組(NP組),30例URSA早孕患者為URSA組;采用Ficoll密度梯度離心法從研究對(duì)象的外周血中分離單個(gè)核細(xì)胞(peripheral blood mononuclear cell,PBMC),流式細(xì)胞分析技術(shù)(Flow cytometry,FCM)檢測2組孕婦外周血中CD14~+單核細(xì)胞(monocyte,MC)上Tim-3、PD-1的表達(dá)水平;酶聯(lián)免疫吸附法(Enzyme linked immunosorbent assay,ELISA)檢測外周血漿中IL-4、IL-6、IL-10、IL-12、IFN-γ的含量;從兩組中各選取10例通過免疫磁珠技術(shù)陽性分選CD14~+單核細(xì)胞,然后分別加入anti-Tim-3、anti-PD-1或anti Tim-3+anti-PD-1阻斷后,給予LPS/R848(5ug/m L)刺激48小時(shí)后流式細(xì)胞技術(shù)檢測CD14~+單核細(xì)胞上PD-1或Tim-3表達(dá)水平及細(xì)胞內(nèi)IL-12分泌情況,采用Graph Pad Prism5.0統(tǒng)計(jì)軟件處理相關(guān)數(shù)據(jù)。結(jié)果:1.與NP組相比,URSA組患者外周血CD14~+MC上Tim-3、PD-1表達(dá)降低(P0.05);相關(guān)性分析Tim-3和PD-1呈正相關(guān)(P0.01)。2.ELISA檢測結(jié)果分析:URSA組外周血漿中IL-4、IL-10濃度較NP組明顯降低(P0.01);IFN-γ、IL-6水平升高(P0.01);MC相關(guān)細(xì)胞因子IL-12在URSA組表達(dá)水平增高,IL-12與Th2型細(xì)胞因子(IL-4、IL-10)呈負(fù)相關(guān)(r=-0.6714,r=-0.5593,P0.01);IL-12與Th1型細(xì)胞因子(IL-6、IFN-γ)呈正相關(guān)(r=0.7740,r=0.5590,P0.01)。3.Tim-3和PD-1與細(xì)胞因子相關(guān)性分析:Tim-3和PD-1與IL-4、IL-10呈正相關(guān)(r=0.6343/0.426,r=0.5356/0.4529,P0.05);與IL-6、IFN-γ呈負(fù)相關(guān)(r=-0.5153/-0.5649,P0.05,r=-0.4518/-0.6543,P0.01),與IL-12呈負(fù)相關(guān)(r=-0.5216,r=-0.540,P0.01)。4.阻斷Tim-3通路后,NP組和URSA組外周血CD14~+MC上PD-1表達(dá)水平降低;URSA組CD14~+MC上PD-1表達(dá)水平低于正常早孕組;同樣,體外阻斷PD-1信號(hào)后,兩組CD14~+MC上Tim-3表達(dá)含量降低,URSA組低于正常早孕組(P0.01)。5.與正常早孕相比,URSA組外周血CD14~+MC內(nèi)IL-12含量增加;阻斷Tim-3或PD-1后,兩組患者外周血CD14~+MC內(nèi)IL-12分泌量增加;在正常早孕組中,阻斷Tim-3較阻斷PD-1后MC內(nèi)IL-12表達(dá)量增加,而在URSA組中,單一阻斷Tim-3或PD-1,CD14~+MC內(nèi)IL-12分泌水平變化無明顯差異(P=0.0515),兩組同時(shí)阻斷Tim-3和PD-1后,CD14~+MC內(nèi)IL-12含量較單獨(dú)阻斷Tim-3或PD-1時(shí)增高明顯(P0.01)。結(jié)論:1.Tim-3和PD-1參與正常妊娠的維持,CD14~+MC上Tim-3和PD-1表達(dá)失衡可能與URSA的發(fā)生有關(guān)。2.在URSA患者外周血中Th1/Th2平衡向Th1偏移,Tim-3和PD-1可能通過抑制MC對(duì)IL-12的分泌,間接調(diào)節(jié)相關(guān)細(xì)胞因子的表達(dá),介導(dǎo)Th1/Th2平衡向Th1漂移,從而參與URSA的發(fā)生。3.通過體外阻斷Tim-3和PD-1信號(hào),發(fā)現(xiàn)Tim-3和PD-1抑制MC分泌IL-12,從而負(fù)性調(diào)節(jié)MC功能,間接調(diào)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- 緯 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- 緯 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- 緯 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- 緯 IL-6 in the plasma of the control group was significantly lower than that in the NP group. 鍦║RSA緇勮〃杈炬按騫沖楂，

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