Th17細胞在人早孕期母—胎界面功能性調(diào)節(jié)作用
發(fā)布時間：2018-08-18 19:24
【摘要】：生理妊娠類似于同種移植,作為同種移植物的胚胎在母體存活直至分娩,實際上反映母體對胚胎的免疫耐受；母體對胚胎的免疫排斥則導致妊娠失敗。揭示母-胎免疫耐受的確切機制,將對人類自然流產(chǎn)等妊娠疾患的防治具有重要意義；并對移植免疫學和腫瘤免疫學的研究將產(chǎn)生推動作用。 母-胎界面主要包括滋養(yǎng)細胞、蛻膜基質(zhì)細胞、蛻膜腺上皮細胞以及免疫細胞。多年來人們從不同角度研究母-胎界面發(fā)生的生物學事件,以闡述母-胎免疫調(diào)節(jié)機制,其中包括母-胎界面Th2型免疫優(yōu)勢的形成,及調(diào)節(jié)性T細胞(Treg)擴增及功能機制。Th17細胞作為新近研究發(fā)現(xiàn)的一群T輔助細胞亞群,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細胞數(shù)量顯著增加。我們以此為切入點,以闡明母-胎界面Th17細胞的來源及其在母-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細胞亞群增加 目的了解人妊娠期間外周血及母-胎界面Th17細胞比例的變化。 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,采用流式細胞術(shù)分析外周血及蛻膜免疫細胞中Th17細胞的比例。 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細胞；而且早孕婦女外周血Th17細胞數(shù)量顯著增加,隨著妊娠進展,其比例逐漸下降,至晚孕期達非孕期水平。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細胞亞群顯著增加,且顯著高于外周血。 結(jié)論Th17細胞可能參與正常妊娠的維持 第二部分人母-胎界面Th17細胞與Treg細胞的分化發(fā)育 目的解析母-胎界面局部微環(huán)境對Th17細胞分化發(fā)育的影響 方法流式細胞術(shù)分析母-胎界面T細胞的表型。滋養(yǎng)細胞、蛻膜基質(zhì)細胞與蛻膜naive CD4+T細胞間接接觸共培養(yǎng)或者滋養(yǎng)細胞、蛻膜基質(zhì)細胞或滋養(yǎng)細胞與蛻膜基質(zhì)細胞共培養(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細胞。妊娠相關(guān)激素處理蛻膜naiveCD4+T細胞,流式細胞術(shù)分析Th17細胞的比例。 結(jié)果母-胎界面存在naive CD4+T細胞。滋養(yǎng)細胞、DSC分別,或兩者共培養(yǎng)均顯著抑制蛻膜naive CD4+T細胞分化發(fā)育為Th17細胞。用母-胎界面主要功能細胞條件培養(yǎng)液處理蛻膜naive CD4+T細胞顯示,母-胎界面主要功能細胞條件培養(yǎng)液可顯著抑制Thl7細胞的分化,促進Treg細胞的優(yōu)勢分化。妊娠相關(guān)激素不影響Th17細胞的分化。 結(jié)論母-胎界面微環(huán)境有利于naive CD4+T細胞分化發(fā)育為Treg細胞,而不利于分化發(fā)育為Th17細胞,呈現(xiàn)Treg偏移。 第三部分人蛻膜基質(zhì)細胞募集通過分泌CCL2和CCL20外周Th17細胞 目的探討母-胎界面Th17細胞的來源 方法磁珠分選外周血CD4+T細胞,采用滋養(yǎng)細胞、蛻膜基質(zhì)細胞或兩者共培養(yǎng)上清液對Th17細胞進行趨化試驗,流式細胞術(shù)分析趨化至下室的Th17細胞的數(shù)量。 結(jié)果DSC條件培養(yǎng)液募集至下室的Th17細胞數(shù)約為外周的2.7倍；而滋養(yǎng)細胞上清及滋養(yǎng)細胞與DSC直接接觸共培養(yǎng)上清募集至下室的Th17細胞數(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Th17細胞的趨化作用。 結(jié)論蛻膜基質(zhì)細胞通過分泌CCL2募集外周血Th17細胞到達蛻膜局部第四部分人母-胎界面Th17細胞促進滋養(yǎng)細胞增殖及侵襲 目的探討Th17細胞對滋養(yǎng)細胞生物學行為的調(diào)控作用 方法免疫組織化學及免疫細胞化學檢測母-胎界面主要功能細胞IL-17R的表達。磁珠分選naive CD4+T細胞,體外誘導Th17細胞分化,制備Th17細胞培養(yǎng)上清,處理滋養(yǎng)細胞,檢測滋養(yǎng)細胞增殖及侵襲能力,流式細胞術(shù)分析滋養(yǎng)細胞的凋亡 結(jié)果滋養(yǎng)細胞、蛻膜基質(zhì)細胞均表達IL-17R。rhIL-17可以劑量依賴性的方式促進滋養(yǎng)細胞的增殖及侵襲；Th17細胞培養(yǎng)上清亦可促進滋養(yǎng)細胞增殖及侵襲；加入IL-17中和抗體后,可顯著抑制滋養(yǎng)細胞增殖及侵襲能力。Th17細胞培養(yǎng)上清處理滋養(yǎng)細胞后,滋養(yǎng)細胞凋亡率下降,從2.23%±0.15%下降至1.88%±0.42%；加入IL-17中和抗體后,導致滋養(yǎng)的凋亡率恢復升高至2.0%±0.11%。 結(jié)論Th17細胞通過分泌細胞因子IL-17調(diào)控滋養(yǎng)細胞的生物學行為,參與早孕胎盤形成。
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
本文編號：2190443
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前2條
1 陳巧英,李大金,金莉萍,朱曉勇,賀銀燕,王明雁;人早孕期外周及蛻膜CD4~+CD25~+調(diào)節(jié)性T細胞變化[J];現(xiàn)代免疫學;2005年05期
2 黃煜;李大金;;人早孕蛻膜基質(zhì)細胞及免疫活性細胞趨化因子受體CXCR6的表達[J];中華微生物學和免疫學雜志;2006年04期
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