【摘要】：生理妊娠類(lèi)似于同種移植,作為同種移植物的胚胎在母體存活直至分娩,實(shí)際上反映母體對(duì)胚胎的免疫耐受；母體對(duì)胚胎的免疫排斥則導(dǎo)致妊娠失敗。揭示母-胎免疫耐受的確切機(jī)制,將對(duì)人類(lèi)自然流產(chǎn)等妊娠疾患的防治具有重要意義；并對(duì)移植免疫學(xué)和腫瘤免疫學(xué)的研究將產(chǎn)生推動(dòng)作用。 母-胎界面主要包括滋養(yǎng)細(xì)胞、蛻膜基質(zhì)細(xì)胞、蛻膜腺上皮細(xì)胞以及免疫細(xì)胞。多年來(lái)人們從不同角度研究母-胎界面發(fā)生的生物學(xué)事件,以闡述母-胎免疫調(diào)節(jié)機(jī)制,其中包括母-胎界面Th2型免疫優(yōu)勢(shì)的形成,及調(diào)節(jié)性T細(xì)胞(Treg)擴(kuò)增及功能機(jī)制。Th17細(xì)胞作為新近研究發(fā)現(xiàn)的一群T輔助細(xì)胞亞群,參與多種自身免疫性疾病及移植物抗宿主病的發(fā)生,我們研究發(fā)現(xiàn)人早孕期間蛻膜Th17細(xì)胞數(shù)量顯著增加。我們以此為切入點(diǎn),以闡明母-胎界面Th17細(xì)胞的來(lái)源及其在母-胎免疫調(diào)節(jié)中的作用。 第一部分人早孕期母-胎界面及外周Th17細(xì)胞亞群增加 目的了解人妊娠期間外周血及母-胎界面Th17細(xì)胞比例的變化。 方法收集正常早、中、晚孕婦女以及正常非孕育齡期婦女外周血,正常早孕婦女蛻膜組織以及非孕婦女子宮內(nèi)膜組織,采用流式細(xì)胞術(shù)分析外周血及蛻膜免疫細(xì)胞中Th17細(xì)胞的比例。 結(jié)果妊娠期婦女外周血、母-胎界面以及正常非孕婦女外周血、子宮內(nèi)膜中均存在Thl7細(xì)胞；而且早孕婦女外周血Th17細(xì)胞數(shù)量顯著增加,隨著妊娠進(jìn)展,其比例逐漸下降,至晚孕期達(dá)非孕期水平。與正常非孕婦女子宮內(nèi)膜組織相比,早孕婦女蛻膜組織Th17細(xì)胞亞群顯著增加,且顯著高于外周血。 結(jié)論Th17細(xì)胞可能參與正常妊娠的維持 第二部分人母-胎界面Th17細(xì)胞與Treg細(xì)胞的分化發(fā)育 目的解析母-胎界面局部微環(huán)境對(duì)Th17細(xì)胞分化發(fā)育的影響 方法流式細(xì)胞術(shù)分析母-胎界面T細(xì)胞的表型。滋養(yǎng)細(xì)胞、蛻膜基質(zhì)細(xì)胞與蛻膜naive CD4+T細(xì)胞間接接觸共培養(yǎng)或者滋養(yǎng)細(xì)胞、蛻膜基質(zhì)細(xì)胞或滋養(yǎng)細(xì)胞與蛻膜基質(zhì)細(xì)胞共培養(yǎng)體系培養(yǎng)上清存在下,采用anti-CD3抗體和anti-CD28抗體活化磁珠分選的母-胎界面naive CD4+T細(xì)胞。妊娠相關(guān)激素處理蛻膜naiveCD4+T細(xì)胞,流式細(xì)胞術(shù)分析Th17細(xì)胞的比例。 結(jié)果母-胎界面存在naive CD4+T細(xì)胞。滋養(yǎng)細(xì)胞、DSC分別,或兩者共培養(yǎng)均顯著抑制蛻膜naive CD4+T細(xì)胞分化發(fā)育為T(mén)h17細(xì)胞。用母-胎界面主要功能細(xì)胞條件培養(yǎng)液處理蛻膜naive CD4+T細(xì)胞顯示,母-胎界面主要功能細(xì)胞條件培養(yǎng)液可顯著抑制Thl7細(xì)胞的分化,促進(jìn)Treg細(xì)胞的優(yōu)勢(shì)分化。妊娠相關(guān)激素不影響Th17細(xì)胞的分化。 結(jié)論母-胎界面微環(huán)境有利于naive CD4+T細(xì)胞分化發(fā)育為T(mén)reg細(xì)胞,而不利于分化發(fā)育為T(mén)h17細(xì)胞,呈現(xiàn)Treg偏移。 第三部分人蛻膜基質(zhì)細(xì)胞募集通過(guò)分泌CCL2和CCL20外周Th17細(xì)胞 目的探討母-胎界面Th17細(xì)胞的來(lái)源 方法磁珠分選外周血CD4+T細(xì)胞,采用滋養(yǎng)細(xì)胞、蛻膜基質(zhì)細(xì)胞或兩者共培養(yǎng)上清液對(duì)Th17細(xì)胞進(jìn)行趨化試驗(yàn),流式細(xì)胞術(shù)分析趨化至下室的Th17細(xì)胞的數(shù)量。 結(jié)果DSC條件培養(yǎng)液募集至下室的Th17細(xì)胞數(shù)約為外周的2.7倍；而滋養(yǎng)細(xì)胞上清及滋養(yǎng)細(xì)胞與DSC直接接觸共培養(yǎng)上清募集至下室的Th17細(xì)胞數(shù)分別是外周的1.1倍及1.8倍。而在DSC培養(yǎng)上清液中加入anti-CCL2中和性抗體后,可顯著抑制DSC培養(yǎng)上清對(duì)Th17細(xì)胞的趨化作用。 結(jié)論蛻膜基質(zhì)細(xì)胞通過(guò)分泌CCL2募集外周血Th17細(xì)胞到達(dá)蛻膜局部第四部分人母-胎界面Th17細(xì)胞促進(jìn)滋養(yǎng)細(xì)胞增殖及侵襲 目的探討Th17細(xì)胞對(duì)滋養(yǎng)細(xì)胞生物學(xué)行為的調(diào)控作用 方法免疫組織化學(xué)及免疫細(xì)胞化學(xué)檢測(cè)母-胎界面主要功能細(xì)胞IL-17R的表達(dá)。磁珠分選naive CD4+T細(xì)胞,體外誘導(dǎo)Th17細(xì)胞分化,制備Th17細(xì)胞培養(yǎng)上清,處理滋養(yǎng)細(xì)胞,檢測(cè)滋養(yǎng)細(xì)胞增殖及侵襲能力,流式細(xì)胞術(shù)分析滋養(yǎng)細(xì)胞的凋亡 結(jié)果滋養(yǎng)細(xì)胞、蛻膜基質(zhì)細(xì)胞均表達(dá)IL-17R。rhIL-17可以劑量依賴(lài)性的方式促進(jìn)滋養(yǎng)細(xì)胞的增殖及侵襲；Th17細(xì)胞培養(yǎng)上清亦可促進(jìn)滋養(yǎng)細(xì)胞增殖及侵襲；加入IL-17中和抗體后,可顯著抑制滋養(yǎng)細(xì)胞增殖及侵襲能力。Th17細(xì)胞培養(yǎng)上清處理滋養(yǎng)細(xì)胞后,滋養(yǎng)細(xì)胞凋亡率下降,從2.23%±0.15%下降至1.88%±0.42%；加入IL-17中和抗體后,導(dǎo)致滋養(yǎng)的凋亡率恢復(fù)升高至2.0%±0.11%。 結(jié)論Th17細(xì)胞通過(guò)分泌細(xì)胞因子IL-17調(diào)控滋養(yǎng)細(xì)胞的生物學(xué)行為,參與早孕胎盤(pán)形成。
[Abstract]:Physiological pregnancy is similar to allogeneic transplantation. Embryos as allografts survive in the mother until childbirth, which in fact reflects the maternal immune tolerance to the embryo; maternal immune rejection of the embryo leads to pregnancy failure. It will also promote the study of transplantation immunology and tumor immunology.
The maternal-fetal interface mainly includes trophoblasts, decidual stromal cells, decidual gland epithelial cells and immune cells. Over the years, biological events at the maternal-fetal interface have been studied from different perspectives to elucidate the mechanisms of maternal-fetal immune regulation, including the formation of Th2 immunodominance at the maternal-fetal interface, and the expansion and function of regulatory T cells (Treg). Mechanisms. Th17 cells, as a subset of T helper cells found recently, are involved in many autoimmune diseases and graft versus host disease. We found that the number of Th17 cells in human decidua increases significantly during early pregnancy. The role of.
The first part is the increase of maternal fetal interface and peripheral Th17 cell subsets in early pregnancy.
Objective to investigate the ratio of Th17 cells in peripheral blood and maternal fetal interface during pregnancy.
Methods Peripheral blood, decidual tissue and endometrial tissue were collected from normal early, middle and late pregnant women and normal non-pregnant women. The proportion of Th17 cells in peripheral blood and decidual immune cells was analyzed by flow cytometry.
Results Thl7 cells were found in peripheral blood, maternal-fetal interface and normal non-pregnant women's endometrium during pregnancy, and the number of Thl7 cells in peripheral blood of early pregnant women increased significantly. With the progress of pregnancy, the proportion of Thl7 cells decreased gradually and reached the level of non-pregnant women's endometrium at the late pregnancy. The Th17 cell subsets of female Decidua Tissue increased significantly, and was significantly higher than that of peripheral blood.
Conclusion Th17 cells may be involved in the maintenance of normal pregnancy.
The second part is the differentiation and development of Th17 cells and Treg cells at the maternal fetal interface.
Objective to analyze the effect of maternal fetal interface microenvironment on differentiation and development of Th17 cells.
Methods The phenotype of T cells at maternal-fetal interface was analyzed by flow cytometry. The magnetic beads were activated by anti-CD3 antibody and anti-CD28 antibody in the presence of the supernatant of the co-culture system of decidual stromal cells, decidual stromal cells and decidual naive CD4 + T cells, decidual stromal cells and decidual stromal cells. Nave CD4+T cells were selected from the maternal-fetal interface. Pregnancy-related hormones were used to treat naive CD4+T cells in decidua. The proportion of Th17 cells was analyzed by flow cytometry.
Results There were naive CD4+T cells in the maternal-fetal interface. Trophoblasts, DSCs, or co-cultures significantly inhibited the differentiation and development of decidual naive CD4+T cells into Th17 cells. Cell differentiation promotes the differentiation of Treg cells. Pregnancy related hormones do not affect the differentiation of Th17 cells.
Conclusion Maternal-fetal microenvironment is beneficial to the differentiation and development of naive CD4+T cells into Treg cells, but not to Th17 cells, showing Treg migration.
In the third part, human decidual stromal cells are recruited by secreting CCL2 and CCL20 peripheral Th17 cells.
Objective to investigate the origin of Th17 cells in maternal fetal interface.
Methods Peripheral blood CD4+T cells were sorted by magnetic beads. Th17 cells were chemotaxis by trophoblast, decidual stromal cells or co-culture supernatant. The number of Th17 cells chemotaxis to the lower chamber was analyzed by flow cytometry.
Results The number of Th17 cells in DSC conditioned medium was about 2.7 times that of peripheral cells, while the number of Th17 cells in supernatant and co-culture supernatant was 1.1 times and 1.8 times that of peripheral cells, respectively. The chemotaxis effect of supernatant on Th17 cells.
Conclusion Decidual stromal cells can promote the proliferation and invasion of trophoblasts by secreting CCL2 to recruit Th17 cells from peripheral blood to the fourth part of human maternal-fetal interface.
Objective to investigate the regulation of Th17 cells on the biological behavior of trophoblast cells.
Methods The expression of IL-17R was detected by immunohistochemistry and immunocytochemistry. Nave CD4+T cells were sorted by magnetic beads, and Th17 cells were induced to differentiate in vitro. The culture supernatant of Th17 cells was prepared. Trophoblasts were treated. The proliferation and invasiveness of trophoblasts were detected. The apoptosis of trophoblasts was analyzed by flow cytometry.
Results Both trophoblasts and decidual stromal cells expressed IL-17R.rhIL-17 in a dose-dependent manner, and the supernatant of Th17 cells also promoted the proliferation and invasion of trophoblasts. The apoptotic rate of trophoblasts decreased from 2.23%+0.15% to 1.88%+0.42% and the apoptotic rate of trophoblasts recovered to 2.0%+0.11% after adding IL-17 neutralizing antibody.
Conclusion Th17 cells regulate the biological behavior of trophoblasts by secreting cytokine IL-17 and participate in placenta formation in early pregnancy.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R392

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