母胎界面炎症效应在妊娠及其相关疾病中的机制研究

发布时间：2018-04-13 10:53

本文选题：白细胞介素27 + 滋养细胞(HTR8/SVneo)　；参考：《重庆医科大学》2016年博士论文

【摘要】：目的:(1)明确白细胞介素27(Interleukin 27,IL-27)及其特异性受体WSX-1在正常妊娠和子痫前期(Preeclampsia,PE)病人中是否存在差异表达;(2)研究IL-27刺激滋养细胞后所发生的炎症效应;(3)探讨IL-27及其下游信号通路在子痫前期发病机制中的可能作用,为子痫前期的治疗提供潜在新靶点和理论基础。方法:(1)ELISA检测IL-27在正常妊娠和PE病人的血清中是否存在差异表达;(2)免疫组化法检测IL-27及其特异性受体WSX-1在正常妊娠和PE病人的胎盘组织中是否存在差异表达;(3)采用RT-PCR和Western Blotting定量检测IL-27的受体WSX-1和gp130在滋养细胞HTR8/SVneo中的表达情况;(4)在滋养细胞HTR8/SVneo中采用RT-PCR筛查IL-27作用的下游炎症因子;(5)采用RT-PCR和Western Blotting分别在RNA和蛋白水平定量验证IL-27作用的下游靶向炎症因子;(6)使用ELISA筛查与IL-27协同作用的经典炎症因子;(7)筛查IL-27作用的下游信号通路,并用Western Blotting在蛋白水平加以验证。结果:(1)正常妊娠和PE病人的血清中IL-27的表达没有差异;(2)免疫组化法证实IL-27及其特异性受体WSX-1能在胎盘组织中正常表达,且PE组较正常对照组表达增加;(3)IL-27的受体WSX-1和gp130在滋养细胞HTR8/SVneo中均有表达;(4)在滋养细胞HTR8/SVneo中IL-27刺激下游炎症因子CXCL10和IL-6的释放呈时间依赖效应;(5)TNF-α能够明显加强由IL-27引起的CXCL10和IL-6的释放;(6)IL-27主要通过激活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)信号通路上调下游炎症因子CXCL10和IL-6。结论:(1)IL-27通过调控滋养细胞释放炎症介质CXCL10和IL-6可触发过度性炎症反应从而参与PE的发生;(2)TNF-α能与IL-27协同作用引起过度性炎症反应;(3)IL-27主要通过激活PI3K-AKT,p38MAPK和JAK/STAT信号通路引起CXCL10和IL-6的释放,致使过度性炎症反应的发生;(4)分别使用PI3K-AKT,p38MAPK和JAK/STAT信号通路的特异性抑制剂LY294002,SB253580和AG490可明显缓解由IL-27刺激引起的过度性炎症效应,从而可能为子痫前期的治疗提供新的思路和靶点。目的:(1)比较足月分娩未启动(term not in labour,TNL)和足月分娩启动(term in labour,TL)时全层胎膜(包括羊膜,绒毛膜和蜕膜)组织匀浆及其上清对外周总白细胞的趋化效应;(2)分别筛选TNL和TL胎膜组织匀浆及其上清中引起白细胞趋化的趋化因子;(3)对靶向趋化因子的趋化能力进行特异性鉴定,挑选出最终激活的趋化因子,为早产的诊治提供潜在的新靶点。方法:(1)建立培养胎膜组织的体外模型;(2)用LMA(Leukocyte migration assay)比较TNL和TL胎膜组织匀浆及上清的趋化活性,并使用特异性抗体标记趋化后的外周白细胞经流式细胞仪检测后予以白细胞亚型分类,并在TNL和TL之间进行比较;(3)采用Proteome Profiler分别筛选TNL和TL胎膜组织匀浆及上清中的激活趋化因子;(4)Quantitative Real-Time PCR(q RT-PCR)和Bio-PlexTM分别从m RNA和蛋白水平验证激活的趋化因子;(5)Bio-PlexTM定量检测TNL和TL病人外周血清中靶向趋化因子的表达水平;(6)LMA验证使用生理剂量的靶向趋化因子的人重组蛋白是否具有趋化活性,及其特异性中和抗体能否减弱这种趋化活性。结果:(1)成功建立了胎膜体外培养模型;(2)经胎膜匀浆及胎膜上清刺激后TL孕妇较TNL孕妇迁移的外周总白细胞数量增加,特别是粒细胞和单核细胞(P0.05),但白细胞亚型比重没有发现明显差异(P0.05);(3)经Proteome Profiler初步筛选与分娩相关的激活趋化因子包括CXCL1,CXCL8,CXCL10,CCL2,CCL5和CCL21;(4)RNA和蛋白水平的定量检测确认CXCL1,CXCL8和CCL21在TNL和TL孕妇胎膜匀浆及上清表达中存在统计学差异(P0.05);(5)比较TNL和TL孕妇血清中这些靶向趋化因子的的表达水平并未发现明显差异(P0.05);(6)TL病人外周总白细胞能够被与上清浓度相似的人重组CXCL1和CXCL8蛋白趋化并呈明显的剂量依赖效应(P0.05),分别加入CXCL1和CXCL8中和抗体后能够明显抑制此趋化效应(P0.05),而在胎膜组织匀浆中未观察到该现象(P0.05)。结论:(1)成功建立了模拟分娩启动时胎膜介导外周白细胞趋化的体外模型,有助于未来对分娩生理机制及早产相关机理的研究;(2)证实分娩启动时胎膜趋化白细胞的数量明显增加,特别是粒细胞和单核细胞,可能在分娩启动的过程中发挥重要作用;(3)经过全面系统地筛查证实由胎膜分泌的CXCL1和CXCL8在介导白细胞浸润胎膜组织触发分娩启动的过程中发挥重要作用,有望成为未来早产诊治的新靶点;(4)胎膜组织中仍存在除CXCL1和CXCL8之外的某些未知的趋化因素参与了分娩的启动。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R714.244

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