本文選題：白細胞介素27 + 滋養(yǎng)細胞(HTR8/SVneo)�。� 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：目的:(1)明確白細胞介素27(Interleukin 27,IL-27)及其特異性受體WSX-1在正常妊娠和子癇前期(Preeclampsia,PE)病人中是否存在差異表達;(2)研究IL-27刺激滋養(yǎng)細胞后所發(fā)生的炎癥效應(yīng);(3)探討IL-27及其下游信號通路在子癇前期發(fā)病機制中的可能作用,為子癇前期的治療提供潛在新靶點和理論基礎(chǔ)。方法:(1)ELISA檢測IL-27在正常妊娠和PE病人的血清中是否存在差異表達;(2)免疫組化法檢測IL-27及其特異性受體WSX-1在正常妊娠和PE病人的胎盤組織中是否存在差異表達;(3)采用RT-PCR和Western Blotting定量檢測IL-27的受體WSX-1和gp130在滋養(yǎng)細胞HTR8/SVneo中的表達情況;(4)在滋養(yǎng)細胞HTR8/SVneo中采用RT-PCR篩查IL-27作用的下游炎癥因子;(5)采用RT-PCR和Western Blotting分別在RNA和蛋白水平定量驗證IL-27作用的下游靶向炎癥因子;(6)使用ELISA篩查與IL-27協(xié)同作用的經(jīng)典炎癥因子;(7)篩查IL-27作用的下游信號通路,并用Western Blotting在蛋白水平加以驗證。結(jié)果:(1)正常妊娠和PE病人的血清中IL-27的表達沒有差異;(2)免疫組化法證實IL-27及其特異性受體WSX-1能在胎盤組織中正常表達,且PE組較正常對照組表達增加;(3)IL-27的受體WSX-1和gp130在滋養(yǎng)細胞HTR8/SVneo中均有表達;(4)在滋養(yǎng)細胞HTR8/SVneo中IL-27刺激下游炎癥因子CXCL10和IL-6的釋放呈時間依賴效應(yīng);(5)TNF-α能夠明顯加強由IL-27引起的CXCL10和IL-6的釋放;(6)IL-27主要通過激活phosphatidylinositol 3-OH kinase-AKT(PI3K-AKT),p38 mitogen-activated protein kinases(p38MAPK)和Janus kinase and a Signal Transducer and Activator of Transcription(JAK/STAT)信號通路上調(diào)下游炎癥因子CXCL10和IL-6。結(jié)論:(1)IL-27通過調(diào)控滋養(yǎng)細胞釋放炎癥介質(zhì)CXCL10和IL-6可觸發(fā)過度性炎癥反應(yīng)從而參與PE的發(fā)生;(2)TNF-α能與IL-27協(xié)同作用引起過度性炎癥反應(yīng);(3)IL-27主要通過激活PI3K-AKT,p38MAPK和JAK/STAT信號通路引起CXCL10和IL-6的釋放,致使過度性炎癥反應(yīng)的發(fā)生;(4)分別使用PI3K-AKT,p38MAPK和JAK/STAT信號通路的特異性抑制劑LY294002,SB253580和AG490可明顯緩解由IL-27刺激引起的過度性炎癥效應(yīng),從而可能為子癇前期的治療提供新的思路和靶點。目的:(1)比較足月分娩未啟動(term not in labour,TNL)和足月分娩啟動(term in labour,TL)時全層胎膜(包括羊膜,絨毛膜和蛻膜)組織勻漿及其上清對外周總白細胞的趨化效應(yīng);(2)分別篩選TNL和TL胎膜組織勻漿及其上清中引起白細胞趨化的趨化因子;(3)對靶向趨化因子的趨化能力進行特異性鑒定,挑選出最終激活的趨化因子,為早產(chǎn)的診治提供潛在的新靶點。方法:(1)建立培養(yǎng)胎膜組織的體外模型;(2)用LMA(Leukocyte migration assay)比較TNL和TL胎膜組織勻漿及上清的趨化活性,并使用特異性抗體標記趨化后的外周白細胞經(jīng)流式細胞儀檢測后予以白細胞亞型分類,并在TNL和TL之間進行比較;(3)采用Proteome Profiler分別篩選TNL和TL胎膜組織勻漿及上清中的激活趨化因子;(4)Quantitative Real-Time PCR(q RT-PCR)和Bio-PlexTM分別從m RNA和蛋白水平驗證激活的趨化因子;(5)Bio-PlexTM定量檢測TNL和TL病人外周血清中靶向趨化因子的表達水平;(6)LMA驗證使用生理劑量的靶向趨化因子的人重組蛋白是否具有趨化活性,及其特異性中和抗體能否減弱這種趨化活性。結(jié)果:(1)成功建立了胎膜體外培養(yǎng)模型;(2)經(jīng)胎膜勻漿及胎膜上清刺激后TL孕婦較TNL孕婦遷移的外周總白細胞數(shù)量增加,特別是粒細胞和單核細胞(P0.05),但白細胞亞型比重沒有發(fā)現(xiàn)明顯差異(P0.05);(3)經(jīng)Proteome Profiler初步篩選與分娩相關(guān)的激活趨化因子包括CXCL1,CXCL8,CXCL10,CCL2,CCL5和CCL21;(4)RNA和蛋白水平的定量檢測確認CXCL1,CXCL8和CCL21在TNL和TL孕婦胎膜勻漿及上清表達中存在統(tǒng)計學(xué)差異(P0.05);(5)比較TNL和TL孕婦血清中這些靶向趨化因子的的表達水平并未發(fā)現(xiàn)明顯差異(P0.05);(6)TL病人外周總白細胞能夠被與上清濃度相似的人重組CXCL1和CXCL8蛋白趨化并呈明顯的劑量依賴效應(yīng)(P0.05),分別加入CXCL1和CXCL8中和抗體后能夠明顯抑制此趨化效應(yīng)(P0.05),而在胎膜組織勻漿中未觀察到該現(xiàn)象(P0.05)。結(jié)論:(1)成功建立了模擬分娩啟動時胎膜介導(dǎo)外周白細胞趨化的體外模型,有助于未來對分娩生理機制及早產(chǎn)相關(guān)機理的研究;(2)證實分娩啟動時胎膜趨化白細胞的數(shù)量明顯增加,特別是粒細胞和單核細胞,可能在分娩啟動的過程中發(fā)揮重要作用;(3)經(jīng)過全面系統(tǒng)地篩查證實由胎膜分泌的CXCL1和CXCL8在介導(dǎo)白細胞浸潤胎膜組織觸發(fā)分娩啟動的過程中發(fā)揮重要作用,有望成為未來早產(chǎn)診治的新靶點;(4)胎膜組織中仍存在除CXCL1和CXCL8之外的某些未知的趨化因素參與了分娩的啟動。
[Abstract]:Objective: (1) clear interleukin 27 (Interleukin 27, IL-27) and its specific receptor WSX-1 in normal pregnancy and pre eclampsia (Preeclampsia, PE) are differentially expressed in patients; (2) the inflammatory effect of IL-27 stimulated trophoblast cells occurred; (3) to investigate the possible role of IL-27 and its downstream the signal pathway in the pathogenesis of preeclampsia, and provide potential new targets and the theoretical basis for the treatment of preeclampsia. Methods: (1) ELISA IL-27 detection in the serum of normal pregnancy and PE patients in the presence of differential expression; (2) whether the difference of expression of IL-27 was detected by immunohistochemical method and its specific receptor WSX-1 in patients with normal pregnancy and PE in placenta; (3) the expression of RT-PCR and Western by Blotting quantitative detection of IL-27 receptor WSX-1 and gp130 in trophoblast cells in HTR8/SVneo; (4) using RT-PCR screening IL- in trophoblast HTR8/SVneo The role of inflammatory cytokines 27; (5) using RT-PCR and Western Blotting downstream target at RNA and protein levels of IL-27 function respectively to the quantitative validation of inflammatory factors; (6) the classic inflammatory cytokines synergize with ELISA screening and IL-27; (7) the downstream signal pathway of IL-27 screening effect, and verified in protein the level of Western Blotting. Results: (1) there is no difference between the expression of serum IL-27 and PE in patients with normal pregnancy; (2) immunohistochemistry confirmed that IL-27 and its specific receptor WSX-1 expression in placenta, and the PE group compared to the normal control group was increased; (3) IL-27 and WSX-1 receptor gp130 HTR8/SVneo was expressed in trophoblast cells; (4) HTR8/SVneo IL-27 in trophoblast cells to stimulate downstream inflammatory factors CXCL10 and IL-6 release in a time dependent effect; (5) TNF- alpha can significantly strengthen by IL-27 induced CXCL10 and IL-6 release; (6) IL-27 Through the activation of phosphatidylinositol 3-OH kinase-AKT (PI3K-AKT), p38 mitogen-activated protein kinases (p38MAPK) and Janus kinase and a Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway up-regulated downstream inflammatory factor CXCL10 and IL-6. conclusion: (1) IL-27 can trigger an excessive inflammatory response which is involved in the development of PE through the regulation of trophoblast cells to release inflammatory medium CXCL10 and IL-6; (2) TNF- alpha and IL-27 synergistic effects caused by excessive inflammatory reaction; (3) IL-27 mainly through the activation of PI3K-AKT, p38MAPK and JAK/STAT signal pathway induced by CXCL10 and IL-6 release, resulting in excessive inflammatory reaction; (4) using PI3K-AKT, a specific inhibitor of LY294002 p38MAPK and JAK/STAT the signal pathway, SB253580 and AG490 can significantly relieve the excessive inflammatory stimulation effect caused by IL-27, and may be from the treatment of preeclampsia Provide new ideas and targets. Objective: (1) comparison of term delivery (term not in labour did not start, TNL) and full-term delivery (term in labour, starting TL) when all layers of membranes (including the amnion, chorion and decidua) chemotaxis of homogenate and supernatant of peripheral white blood cells of the total; (2). The screening of leukocyte chemotactic chemokines induced by homogenate supernatant of TNL and TL and fetal membranes; (3) to target specific identification to chemokine chemotactic ability, pick out the final activation of chemokines, provide a potential new target for the diagnosis and treatment of preterm birth. Methods: (1) the establishment of an in vitro model of fetal tissue culture; (2) with LMA (Leukocyte migration assay) TNL TL and supernatant of homogenate and fetal tissue chemotactic activity, and the use of specific antibody to more peripheral white blood cells was detected by flow cytometry to leukocyte subtype classification, and between TNL and TL Comparison; (3) by Proteome Profiler. The screening of chemokines TNL and TL activation of homogenate and supernatant of fetal membranes; (4) Quantitative Real-Time PCR (Q RT-PCR) and Bio-PlexTM respectively from m RNA and protein expression levels of chemokine activation; (5) targeting expression of chemokines in serum quantitative detection of Bio-PlexTM TNL and TL in patients with peripheral; (6) LMA verification using physiological doses of targeting chemokines by human recombinant protein with chemotactic activity and specific neutralizing antibody can attenuate the chemotactic activity. Results: (1) successfully established in vitro model of cultured fetal membranes (2); increase the number of membranes and membrane homogenate was stimulated by TL in pregnant women than TNL pregnant women migration total peripheral white blood cells, especially neutrophils and monocytes (P0.05), but the proportion of white blood cell subtypes found no significant difference between (P0.05); (3) by Proteome Profiler and preliminary screening Chemokines including CXCL1, CXCL8, CXCL10, CCL2 and CCL5 activation associated with childbirth, CCL21; (4) quantitative detection of RNA and protein levels of CXCL1 and CCL21 CXCL8 confirmed that there were significant differences in the expression of TNL and TL in fetal membrane homogenate and supernatant (P0.05); (5) the target comparison of TNL and TL in pregnant women the serum to the expression level of chemokines did not find significant difference (P0.05); (6) TL patients with total peripheral white cells can be with supernatant of similar concentrations of human recombinant CXCL1 and CXCL8 proteins of chemotaxis and showed a significant dose dependent effect (P0.05), were added to CXCL1 and CXCL8 neutralizing antibody to this significantly inhibited chemotaxis (P0.05), and in the fetal tissue homogenate was not observed this phenomenon (P0.05). Conclusion: (1) successfully established a simulation of membrane mediated delivery starting chemotaxis in vitro model of peripheral white blood cells, contribute to the future of childbirth premature physiological mechanism and related mechanism Study; (2) confirmed delivery starting when the number of white blood cell membranes more significantly increased, especially granulocytes and monocytes may play an important role in the process of parturition; (3) after play an important role in comprehensive and systematic screening confirmed by secreting CXCL1 and CXCL8 membranes in mediating leukocyte infiltration fetal tissue triggers the parturition process, is expected to become a new target for future diagnosis and treatment of premature delivery; (4) some unknown except CXCL1 and CXCL8 still exist in fetal membranes of chemotactic factors involved in the delivery of start.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R714.244

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