【作者】 姚穗；

【导师】 周昌菊；

【作者基本信息】 中南大学 ， 妇产科学， 2007， 博士

【摘要】 早产是常见的妊娠并发症，约占整个妊娠的5％-15％。早产儿发病率和病死率明显高于正常足月产新生儿，约占围产儿死亡率的2／3，因此一直受到产科界的关注。研究发现早产与宫内感染关系密切，我们将与宫内感染相关的早产称为感染性早产。根据报道早产孕妇中有约58％-88％被证实有组织学绒毛膜羊膜炎改变。但临床上多数孕妇呈现亚临床表现，症状不典型，给临床诊断带来困难。以往感染性早产诊断主要依靠羊水细菌培养阳性或胎盘胎膜组织学检查发现绒毛膜羊膜炎，但上述方法前者所需时间较长，后者需要产后才能确诊，不能达到早期诊断早期治疗的目的。近年来有研究者通过检测孕妇外周血中CRP(c-reactive protein，CRP)、补体及免疫球蛋白ⅠgM、ⅠgG、ⅠgA、ⅠgS等预测感染性早产，但临床应用中发现这些指标均存在敏感性和特异性较低的问题。因此从早产发病机制着手，寻找早期反映羊膜腔内感染敏感性和特异性高的指标成为目前研究的热点。NF-κB是近年来发现的一种核转录因子，启动并调节机体炎症和免疫反应、凋亡及细胞的增值分化等相关基因的转录。参与早产及分娩发动过程的许多炎性因子如IL-8等的分泌，均受NF-κB的调控。也有报道MMP-9的基因上游调节元件中含有NF-κB结合位点，与NF-κB结合后启动基因的转录。白介素-8(interleukin-8，IL一8)是一种重要的炎性细胞因子，参与机体的炎性反应，被认为是介导羊膜腔内感染的炎性介质，能客观反映羊膜腔内感染的情况，IL-8的表达明显增高提示宫内感染的存在。根据大量研究推测IL-8可能通过刺激多核白细胞的侵润继而促进MMPs的分泌。MMPs是一组锌依赖的蛋白水解酶，主要参与细胞外基质的代谢。MMP-9是MMPs家族中的一员，可特异性地降解Ⅳ型胶原。Ⅳ胶原主要存在于绒毛膜和羊膜的基底层。研究发现MMP-9活性的升高与胎膜中胶原的降解密切相关，MMP-9参与正常足月分娩胎膜破裂的发生。进一步研究发现伴有羊膜腔内感染的胎膜早破患者，其体内MMP-9的表达明显高于正常妊娠。但MMP-9与感染的关系并未得到阐明。NF-κB是IL-8、MMP-9的基因表达促进因子，IL-8参与促进MMP-9的分泌，MMP-9表达异常增高导致胎膜破裂。NF-κB、IL-8、MMP-9在亚临床绒毛膜羊膜炎的发生中是否存在相关性?它们在孕妇血清、胎盘胎膜中的表达如何，又与胎儿脐血中的表达存在什么关系，目前尚未见报道。因此，本实验选择了早产这一常见的妊娠并发症作为研究对象，从胎盘胎膜入手，通过不同的实验方法观察NF-κB与IL-8及MMP-9的表达变化及组织形态学的改变；比较母外周血及脐血中NF-κB、IL-8与MMP-9水平。从而探讨NF-κB、IL-8与MMP-9的含量与亚临床绒毛膜羊膜炎的关系及其在发病机制中的作用，为早期预测感染性早产及为临床上早期制定治疗方案提供理论依据。NF-κB P65蛋白预测亚临床绒毛膜羊膜炎价值的研究目的：研究NF-κB P65在亚临床绒毛膜羊膜炎孕母血、脐血、胎盘胎膜组织中的表达变化，探讨其在亚临床绒毛膜羊膜炎发病机制中的作用，评价其预测亚临床绒毛膜羊膜炎及亚临床绒毛膜羊膜炎治疗效果的临床价值方法：选择早产临产组36例，先兆早产组14例，足月妊娠自然临产组37例，足月妊娠同期对照组38例，早产同期对照组18例。通过HE染色、免疫组织化学、免疫细胞化学、Western Blotting、RT-PCR五种实验方法，观察NF-κB P65在亚临床绒毛膜羊膜炎孕母血、脐血、胎盘胎膜组织中的表达变化。结果：1．各组孕妇胎盘胎膜组织病理检查结果36例早产临产组中有24例(66.67％)为亚绒毛膜羊膜炎患者，其中轻度12例，中度10例，重度2例；37例足月妊娠自然临产组中有7例(18.92％)为亚绒毛膜羊膜炎患者，其中轻度5例，中度2例，重度0例；38例足月妊娠同期对照组中有3例(7.89％)为绒毛膜羊膜炎患者，其中轻度3例，中度0例，重度0例。2．两组孕妇NF-κB P65在胎盘胎膜组织中表达水平及临床价值2.1免疫组织化学测定两组孕妇NF-κB P65蛋白在胎盘胎膜组织表达比较用免疫组织化学法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇NF-κB P65蛋白表达水平。其的平均染色强度变化妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为8.00(2.00～8.00)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为4.00(2.00～6.00)，两组间比较差异有统计学意义(P＜0.001)。表达比较NF-κB P65在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为48.43(7.75～97.27)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为27.98(7.75～61.83)，两组间比较差异有统计学意义(P＜0.001)。2.3 RT-PCR测定两组孕妇NF-κB P65 mRNA在胎盘胎膜组织表达比较NF-κB P65 mRNA在妊娠合并亚临床绒毛膜羊膜炎组(47.51±17.21)比妊娠无亚临床绒毛膜羊膜炎组(31.30±13.60)高，两组间比较差异有统计学意义(P＜0.001)。3．NF-κB P65在脐血中的表达3.1免疫细胞化学测定两组孕妇脐血白细胞中NF-κB P65蛋白表达比较用免疫细胞化学法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇脐血白细胞中NF-κB P65蛋白表达水平。其的平均染色强度变化在妊娠合并亚临床绒毛膜羊膜炎组中的中位数和范围为4.00(2.00～8.00)，妊娠无亚临床绒毛膜羊膜炎组中的中位数和范围为2.00(2.00～6.00)，两组间比较差异有统计学意义(P＜0.001)。3.2 Western Blotting测定两组孕妇脐血白细胞中NF-κB P65蛋白表达比较NF-κB P65在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为27.38(17.89～69.40)，妊娠无亚临床绒毛膜羊膜炎组中位数和范围为25.18(13.92～32.46)，两组间比较差异有统计学意义(P＜0.05)。3.3 RT-PCR测定各组孕妇脐血自细胞中NF-κB P65 mRNA表达比较NF-κB P65 mRNA在妊娠合并亚临床绒毛膜羊膜炎组中的中位数和范围为30.76(17.79～74.72)，妊娠无亚临床绒毛膜羊膜炎组中的中位数和范围为22.67(13.57～30.12)，两组间比较差异有统计学意义(P＜0.01)。4．NF-κB P65在母血中的表达4.1免疫细胞化学测定两组孕妇母血白细胞中NF-κB P65蛋白表达比较用免疫细胞化学法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇母血白细胞中NF-κB P65蛋白表达水平。其的平均染色强度变化在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为8.00(3.00～8.00)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为4.00(2.00～6.00)，两组间比较差异有统计学意义(P＜0.001)。4.2 Western Blotting测定两组孕妇母血白细胞中NF-κB P65蛋白表达比较NF-κB P65在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为34.22(19.24～88.49)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为31.45(11.33～48.44)，两组间比较差异有统计学意义(P＜0.01)。4.3 RT-PCR测定各组孕妇母血白细胞中NF-κB P65 mRNA表达比较NF-κB P65 mRNA在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为42.58(28.57～81.11)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为23.56(14.92～42.87)，两组间比较差异有统计学意义(P＜0.001)。5胎盘胎膜组织、脐血及母血中NF-κB P65表达的相关性比较5.1产妇胎盘胎膜组织、脐血及母血中NF-κB P65蛋白IHC／ICC法检测结果的相关性比较分析IHC／ICC法检测胎盘胎膜组织、脐血及母血中NF-κB P65蛋白的表达三者间的相互关系，发现胎盘胎膜组织NF-κB P65蛋白的表达与脐血NF-κB P65蛋白的表达存在正相关性，相关系数r=0.498，P＜0.01；胎盘胎膜组织NF-κB P65蛋白的表达与母血中NF-κB P65蛋白的表达存在正相关性，相关系数r=0.581，P＜0.01；脐血NF-κB P65蛋白的表达与母血中NF-κB P65蛋白的表达存在正相关性，相关系数r=0.769，P＜0.01。5.2产妇胎盘胎膜组织、脐血及母血中NF-κB P65蛋白WesternBlotting法检测结果的相关性比较分析Western Blotting法检测胎盘胎膜组织、脐血及母血中NF-κB P65蛋白的表达三者间的相互关系，发现胎盘胎膜组织NF-κB P65蛋白的表达与脐血NF-κB P65蛋白的表达存在正相关性，相关系数r=0.583，P＜0.01；胎盘胎膜组织NF-κB P65蛋白的表达与母血中NF-κB P65蛋白的表达存在正相关性，相关系数r=0.714，P＜0.01；脐血NF-K B P65蛋白的表达与母血中NF-K B P65蛋白的表达存在正相关性，相关系数r=0.802，P＜0.01。5.3产妇胎盘胎膜组织、脐血及母血中NF-κB P65 mRNA RT-PCR法检测结果的相关性比较分析RT-PCR法检测胎盘胎膜组织、脐血及母血中NF-κB P65mRNA的表达三者间的相互关系，发现胎盘胎膜组织NF-κB P65 mRNA的表达与脐血NF-κB P65 mRNA的表达存在正相关性，相关系数r=0.522，P＜0.01；胎盘胎膜组织NF-κB P65mRNA的表达与母血中NF-KB P65 mRNA的表达存在正相关性，相关系数r=0.571，P＜0.01；脐血NF-κB P65 mRNA的表达与母血中NF-κB P65 mRNA的表达存在正相关性，相关系数r=0.892，P＜0.01。6．NF-κB P65的ROC曲线6.1产妇脐血白细胞中NF-κB P65的ROC曲线显示出运用ICC、Western Blotting、RT-PCR三种方法检测脐血白细胞中NF-κB P65的临界点分别为3.50，25.64和22.83，为最适预测有无绒毛膜羊膜炎的值。三种方法曲线下NF-κB P65的积分面积分别为0.728，0.653和0.749，三者面积比较差异有统计学意义(P＜0.05)。其中ICC检测脐血白细胞中NF-κB P65有较好的敏感性(75.8％)，特异性(51.9％)。6.2产妇母血白细胞中NF-κB P65的ROC曲线显示出运用ICC、Western Blotting、RT-PCR三种方法检测母血白细胞中NF-κB P65的临界点分别为5.00，31.61和31.68，为最适预测有无绒毛膜羊膜炎的值。三种方法曲线下NF-κB P65的积分面积分别为0.899，0.676和0.920，三者面积比较差异有统计学意义(P＜0.05)。其中运用RT-PCR法检测母血白细胞中NF-κB P65mRNA有较好的敏感性(87.9％)，特异性(89.3％)。结论：1．亚临床绒毛膜羊膜炎孕母血白细胞中NF-κB P65表达增高，可能作为预测亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价亚临床绒毛膜羊膜炎治疗效果实验室指标之一；2．亚临床绒毛膜羊膜炎胎儿脐血中NF-κB P65表达增高，可能作为预测早产亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价治疗亚临床绒毛膜羊膜炎预后实验室指标之一；3．亚临床绒毛膜羊膜炎孕妇胎盘胎膜组织中NF-κB P65蛋白表达增高，提示NF-κB P65在亚临床绒毛膜羊膜炎发病机制中起到重要的作用。Ik-8预测亚临床绒毛膜羊膜炎价值的研究目的：研究IL-8在亚临床绒毛膜羊膜炎孕妇母血、脐血、胎盘胎膜组织中的表达水平的变化，探讨其在亚临床绒毛膜羊膜炎发病机制中的作用，评价其早期预测感染性早产的价值。方法：选择早产临产组36例，先兆早产组14例，足月妊娠自然临产组37例，足月妊娠同期对照组38例，早产同期对照组18例。通过ELISA、HE染色、免疫组织化学3种实验方法，观察IL-8蛋白在早产亚临床绒毛膜羊膜炎孕妇母血、脐血、胎盘胎膜组织中的表达变化。结果：1．各组孕妇胎盘胎膜组织病理检查结果孕妇胎盘胎膜组织病理检查结果同第一章。2．免疫组织化学测定各组孕妇IL-8蛋白在胎盘胎膜组织表达比较用免疫组织化学法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇IL-8蛋白表达水平。其的平均染色强度变化在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为4.00(2.00～8.00)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为2.00(2.00～6.00)，两组间比较差异有统计学意义(P＜0.001)。3．IL-8在脐血清中的表达用ELISA法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇脐血清IL-8中表达水平。IL-8在妊娠合并亚临床绒毛膜羊膜炎组(33.43±12.98ng／ml)比妊娠无亚临床绒毛膜羊膜炎组(20.21±11.47ng／ml)高，两组间比较差异有统计学意义(P＜0.001)。4．IL-8在母血清中的表达用ELISA法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇母血清IL-8中表达水平。IL-8在妊娠合并亚临床绒毛膜羊膜炎组中的中位数和范围为45.14(25.15～63.21)ng／ml，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为24.18(0.57～65.13)ng／ml，两组间比较差异均有统计学意义(P＜0.001)。5．胎盘胎膜组织、脐血及母血中IL-8表达的相关性比较分析胎盘胎膜组织、脐血及母血中IL-8的表达三者间的相互关系，发现盘胎膜组织IL-8的表达与脐血IL-8的表达存在正相关性，相关系数r=0.296，P＜0.01；胎盘胎膜组织IL-8的表达与母血中IL-8的表达存在正相关性，相关系数r=0.304，P＜0.01；脐血IL-8的表达与母血中IL-8的表达存在正相关性，相关系数r=0.532，P＜0.01。6．IL-8的ROC曲线6.1产妇脐血清中IL-8的ROC曲线显示出脐清中IL-8的临界点为25.40ng／ml，为最适预测有无亚临床绒毛膜羊膜炎的值。曲线下IL-8的积分面积为0.773。其敏感性为85.3％，特异性为70.1％。6.2母血清IL-8的ROC曲线显示出母血清IL-8的临界点为34.76na／m1，为最适预测有无绒毛膜羊膜炎的值。曲线下IL-8的积分面积为0.798。其敏感性为79.4％，特异性为79.2％。结论：1．亚临床绒毛膜羊膜炎孕妇母血清中IL-8表达增高，可能作为预测早产亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价早产亚临床绒毛膜羊膜炎治疗效果实验室指标之一；2．亚临床绒毛膜羊膜炎胎儿脐血中IL-8表达增高，可能作为预测亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价治疗亚临床绒毛膜羊膜炎预后实验室指标之一；3．亚临床绒毛膜羊膜炎孕妇胎盘胎膜组织中IL-8表达增高，提示IL-8因子参与亚临床绒毛膜羊膜炎发病。4．亚临床绒毛膜羊膜炎胎儿脐血中IL-8蛋白表达增高，可能作为预测新生儿感染性疾病的实验室指标之一，有助于早期协助诊断新生儿感染性疾病并及时指导临床治疗；并且可能作为新生儿感染性疾病疗效及愈后评价的实验室指标。MMP-9预测亚临床绒毛膜羊膜炎价值的研究目的：研究MMP-9在亚临床绒毛膜羊膜炎孕妇母血、脐血、胎盘胎膜组织中的表达水平的变化，探讨其在亚临床绒毛膜羊膜炎发病机制中的作用，评价其早期预测感染性早产的价值。方法：选择早产临产组36例，先兆早产组14例，足月妊娠自然临产组37例，足月妊娠同期对照组38例，早产同期对照组18例。通过ELISA、HE染色、免疫组织化学3种实验方法，观察MMP-9蛋白在早产亚临床绒毛膜羊膜炎孕妇母血、脐血、胎盘胎膜组织中的表达变化。结果：1．各组孕妇胎盘胎膜组织病理检查结果孕妇胎盘胎膜组织病理检查结果同第一章。2．免疫组织化学测定各组孕妇MMP-9蛋白在胎盘胎膜组织表达比较用免疫组织化学法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇MMP-9蛋白表达水平。其的平均染色强度变化在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为6.00(2.00～8.00)，妊娠无亚临床绒毛膜羊膜炎组的中位数和范围为2.00(2.00～6.00)，两组间比较差异有统计学意义(P＜0.001)。3．MMP-9在脐血清中的表达用ELISA法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇脐血清MMP-9中表达水平。MMP-9在妊娠合并亚临床绒毛膜羊膜炎组(368.41±110.55ng／m1)比妊娠无亚临床绒毛膜羊膜炎组(281.67±96.73ng／ml)高，两组间比较差异有统计学意义(P＜0.001)。4．MMP-9在母血清中的表达用ELISA法测定妊娠合并亚临床绒毛膜羊膜炎组与妊娠无亚临床绒毛膜羊膜炎组两组孕妇母血清MMP-9中表达水平。MMP-9在妊娠合并亚临床绒毛膜羊膜炎组的中位数和范围为458.19(315.52～522.15)ng／ml，妊娠无亚临床绒毛膜羊膜炎组307.15(181.29～533.30)ng／ml，两组问比较差异有统计学意义(P＜0.001)。5．胎盘胎膜组织、脐血及母血中MMP-9表达的相关性比较分析胎盘胎膜组织、脐血及母血中MMP-9的表达三者间的相互关系，发现胎盘胎膜组织MMP-9的表达与脐血MMP-9的表达存在正相关性，相关系数r=0.420，P＜0.01；胎盘胎膜组织MMP-9的表达与母血中MMP-9的表达存在正相关性，相关系数r=0.412，P＜0.01；脐血MMP-9的表达与母血中MMP-9的表达存在正相关性，相关系数r=0.561，P＜0.01。6．MMP-9的ROC曲线6．1产妇脐血清中MMP-9的ROC曲线显示出脐清中MMP-9的临界点为317.50ng／m1，为最适预测有无亚临床绒毛膜羊膜炎的值。曲线下MMP-9的积分面积为0.720。其敏感性为73.5％，特异性为71.4％。6．2产妇母血清MMP-9的ROC曲线显示出母血清MMP-9的临界点为360.81ng／ml，为最适预测有无亚临床绒毛膜羊膜炎的值。曲线下MMP-9的积分面积为0.823。其敏感性为87.4％，特异性为72.0％。结论：1．亚临床绒毛膜羊膜炎孕妇母血清中MMP-9蛋白表达增高，可能作为预测亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价亚临床绒毛膜羊膜炎治疗效果实验室指标之一；2．亚临床绒毛膜羊膜炎胎儿脐血中MMP-9蛋白表达增高，可能作为预测亚临床绒毛膜羊膜炎实验室指标之一，且有可能作为评价治疗亚临床绒毛膜羊膜炎预后实验室指标之一；3．亚临床绒毛膜羊膜炎孕妇胎盘胎膜组织中MMP-9蛋白表达增高，提示MMP-9蛋白参与亚临床绒毛膜羊膜炎发病。更多还原

【Abstract】 Prematurity is a common complication of pregnancy. The incidence rate is about 5% to 15%.The morbidty and case fatality of premature infant are more than the normal ,which is about 2/3 in perinatal mortality rate . So prematurity is always the focus of Obstetrics. The intrauterine infection is related with premature delivery. About 58 to 88 percent pregnant women of preterm delivery have histological change of chorioamnionitis. But most of pregnant women persent subclinical manifestation,which has not typical clinical symptoms, and great difficulty diagnosis. In the past, the diagnosis of prematurity associated with infection depended on positive outcome of bacterial culture of the amniotic fluid or the histology diagnosis. But both of them could not achieve the purpose of early diagnosis, because the first one took a long time, the later could be later diagnosed after parturition. For the past few years, some researchers predicted preterm delivery by detecting C-reactive pretein(CRP), complement or IgM、IgG、IgA、IgS in maternal blood. But these index had lower sensibility and specificity in clinical application. It is focus for researches begin with the pathogenesy of the preterm delivery that we search the sensitive and specific indexs to reflect amniotic cavity inflection.NF-kappa B is a kind’of nuclear factor, which can start and regulate the transcripion of related gene such as inflammation ,immune reaction ,apoptosis ,cell increase and differentiation.Many inflammatory factor, such as IL-8 and so on who participate the onset course of immature labour and accouchement are regulated by NF-kappa B. It is reported that the up stream regulatory element of MMP-9 gene contain the binding site of NF-kappa B, which can binding NF-kappa B to promote the transcriptionInterleukin-8 is a kind of important inflammatory cytokine, which participate the inflammatory reaction in organism and mediate the amniotic cavity infection and reflect the infection extent objectively. The high level of IL-8 presents the intrauterine infection. A great quantity study presume IL-8 may promote MMPs excrete by stimulating polycyte infiltrating. Matrix metalloproteinases is a family of Zinc dependent proteolytic which can mainly participate the metabolism of extracellular matrix. MMP-9(matrix metalloproteinase, MMP-9) is one of MMPs family which specially degrade typeⅣcollagen. TypeⅣcollagen mainly reside in basal layer of chorion and amnion. The investigation shows that the activity of MMP-9 increased has close relationship with fetal membrane degradation. MMP-9 also participate the development of rupture of membranes in term labor. Further search demonstrate that the level of MMP-9 is obviously higher than normal in premature rupture of membranes(PROM) with amniotic cavity infection. But the relation between MMP-9 and infection is still not clear.NF-κB promote the expression of IL-8or MMP-9. IL-8 enhance the secrete of MMP-9. The abnormal high expression will induce membrane rupture. But if NF-κB, IL-8, MMP-9 are related in development of subclinical chorioamnionitis in preterm delivery? What are they expression in maternal blood ,afterbirth tissue or cord blood? These is no reports about it upto now.So, we select immature labor as the object to research, which is a frequent complication of cyophoria. To observe the expression and changes of histomorphology, and compare the level of NF-κB, IL,8 and MMP-9 in peripheral blood and cord blood of mother.To investigate the contents of NF-κB, IL-8 and MMP-9,whose relationship with subclinical chorioamnionitis and the effect in pathogenesy.It is helpful for offering theory of infective immature labor to estimate and set up therapeutic regimen. Objective: To investigate the change of the p65 submit of the nuclear factor kappa B family in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis, and analyze its effect on the pathogenesis of subclinical Chorioamnionitis , then to estimate its clinical value and therapeutic efficacy in predicting subclinical Chorioamnionitis.Methods: To chose 36 cases of preterm birth patients in labor , 14 of cases threatened premature labor , 37 cases of full term gravida with spontaneous inlabor, 38 cases of full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients. To observe the change of the p65 submit of the nuclear factor kappa B family in maternal blood, umbilical cord blood, placenta and fetal membrane in preterm birth patients with subclinical Chorioamnionitis by five methods,which were HE staining ,immunohistochemical staining , immunocytochemical staining,Western Blotting and RT-PCR.Results:1. The pathematology results of placenta and fetal membrane in each groups:There were 24 cases who were subclinical chorioamnionitis patients in 36 cases ofpreterm birth patients in labor (66.67%) . 12 cases are mild,10 cases are midrange,2 cases are severe among them. There were 7 cases who were subclinical chorioamnionitis patient in 37 cases of the full term gravida with spontaneous inlabor group (18.92%). 5 cases are mild,2cases are midrange,none is severe among them. There were 3 cases who were subclinical chorioamnionitis patient in 38 cases of the full term gravida without threatened labor group (7.89%). 3 cases are mild, none is midrange and severe..2. The expression of NF-κB P65 in placenta and fetal membrane:2.1 Compared the expression of NF-κB P65 in placenta and fetal membrane by IHC in each groups:The expression of NF-κB P65 were detected in placenta and fetal membrane by im_munohistochemistry staining method in 2 groups,which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group.The median and range of the average staining intensity of NF-κB P65 was [8.00 (2.00～8.00) ] in pregnant with subclinical chorioamnionitis group,and [4.00(2.00～6.00)] in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).2.2 Compared the expression of NF-κB P65 in placenta and fetal membrane by Western Blotting in each groups:The median and range of the average staining intensity of NF-κB P65 was 48.43 (7.75～97.27) in pregnant with subclinical chorioamnionitis group, and 27.98 (7.75～61.83)in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).2.3 Compared the expression of NF-κB P65 mRNA in placenta and fetal membrane by RT-PCR in each groups:The average staining intensity of NF-κB P65 was 47.51±17.21 in pregnant with subclinical chorioamnionitis group, and 31.30±13.60 in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).3. The expression of NF-κB P65 in umbilical cord blood in each groups:3.1 The expression of NF-κB P65 in umbilical cord blood by ICC in each groups:The expression of NF-κB P65 were detected in umbilical cord blood by immunocytochemical staining method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 was 4.00 (2.00～8.00) in pregnant with subclinical chorioamnionitis group, and 2.00 (2.00～6.00) in pregnant without subclinical chorioamnionitis group. There was significant difference between the two groups(P＜0.001).3.2 The expression of NF-κB P65 in umbilical cord blood by Wstern Blotting in each groups:The median and range of the expression of NF-κB P65 was 27.38 (17.89～69.40) in pregnant with subclinical chorioamnionitis groupthan, and 25.18 (13.92～32.46) in pregnant without subclinical chorioamnionitis group.There was significant difference between the two groups(P＜0.001).3.3 The expression of NF-κB P65 mRNA in umbilical cord blood by RT-PCR in each groups:The median and range of the expression of NF-κB P65 was30.76 (17.79～74.72) in pregnant with subclinical chorioamnionitis group, and 22.67 (13.57～30.12) in pregnant without subclinical chorioamnionitis group. There was significant difference between the two groups(P＜0.001).4. The expression of NF-κB P65 in maternal blood in each groups:4.1 The expression of NF-κB P65 in maternal blood by ICC in each groups:The expression of NF-κB P65 were detected in maternal blood by immunocytochemical staining method in 2 groups, which included pregnant with subclinical chofioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 in pregnant with subclinical chofioamnionitis group is 8.00 (3.00～8.00), and 4.00 (2.00～6.00) in pregnant without subclinical chofioamnionitis group. There was significant difference between the two groups(P＜0.001).4.2 The expression of NF-κB P65 in maternal blood by Wstern Blotting in each groups:The median and range of the expression of NF-κB P65 was 34.22 (19.24～88.49) in pregnant with subclinical chorioammionitis group, and 31.45 (11.33～48.44) in pregnant without subclinical chorioamnionitis .group.There was significant difference between the two groups(P＜0.001).4.3 The expression of NF-κB P65 mRNA in maternal blood by RT-PCR in each groups:The expression of NF-κB P65 mRNA were detected in maternal blood by RT-PCR method in pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of NF-κB P65 was the higher in pregnant with subclinical chorioamnionitis group[42.58 (28.57～81.11) ] than those in Pregnant without subclinical chorioamnionitis group[23.56 (14.92～42.87) ].There was significant difference between the two groups(P＜0.001).5. The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood:5.1 The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by IHC/ICC:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood ,we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.498,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.581 ,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.769,p＜0.01). 5.2 The correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by Western Blotting:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by Western Blotting, we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.583,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.714,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.803,p＜0.01).5.3 The correlation of NF-KB P65 mRNA in placenta and fetal membrane, umbilical cord blood, maternal blood by RT-PCR:To compare the correlation of NF-κB P65 in placenta and fetal membrane, umbilical cord blood, maternal blood by RT-PCR,, we found that the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.522,p＜0.01); the concentration of NF-κB P65 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.571,p＜0.01); the concentration of NF-κB P65 in umbilical cord blood was positively correlated with that of maternal blood (r=0.892,p＜0.01).6. The ROC curve of NF-κB P656.1 The ROC curve of NF-κB P65 in umbilical cord blood:The critical point of NF-κB P65 in umbilical cord blood were 3.50, 25.64 and 22.83 respectively by ICC、Western Blotting、RT-PCR, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve were 0.728, 0.653 and 0.749 respectively. There was significant difference between any groups of the three (P＜0.05). It was NF-KB P65 tested by ICC that had better sensitivity(79.4%) and specificity(51.9% )for diagnosing chorioamnionitis.6.2 The ROC curve of NF-κB P65 in maternal blood:The critical point of NF-κB P65 in umbilical cord blood were 5.00, 31.61 and 31.68 respectively by ICC、Western Blotting、RT-PCR three methods, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve were 0.899, 0.676 and0.920 respectively. There was significant difference between any groups of the three (P＜0.05). It was NF-KB P65 tested by RT-PCR that had better sensitivity (87.9 %)and specificity (89.3 %)for diagnosing chorioamnionitis.Conclusion:1. The high expression of the p65 submit of the nuclear factor kappa B family in maternal blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical Chorioamnionitis patients and in evaluating the therapeutic effect on patients with subclinical Chorioamnionitis.2. The high expression of the p65 submit of the nuclear factor kappa B family in umbilical cord blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical chorioamnionitis patients and in evaluating the therapeutic effect on patients with subclinical chorioamnionitis.3. It was high expression of the p65 submit of the nuclear factor kappa B family in placenta and fetal membrane in subclinical Chorioamnionitis. which indicates that the p65 submit of the nuclear factor kappa B plays an important part in the pathogenetic of subclinical Chorioamnlonitis patients. Objective: To investigate the change of the interleukin-8 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis patients,, to analyze its effect on the pathogenesis of subclinical Chorioamnionitis , to estimate clinical value in predicting subclinical ChorioamnionitisMethods: To chose 36 cases of preterm birth patients in labor, 14 cases of threatened premature labor, and 37 cases full term gravida with spontaneous in labor, 38 cases full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients. It were enzyme-linked immunosorbentassay, HE staining, immunohistochemical staining that used to observe the change of the interleukin-8 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioanmionitis.Results:1. The pathematology results of placenta and fetal membrane in each groups:The pathematology results of placenta and fetal membrane were the same that of the ChapterⅠ.2. Compared the expression of IL-8 in placenta and fetal membrane by IHC in each groups:The expression of IL-8 were detected in 2 groups by immunohistochemistry staining method, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the average staining intensity of IL-8 was the higher in pregnant with subclinical chorioamnionitis group[4.00 (2.00～8.00)] than those in pregnant without subclinical chorioamnionifis group[2.00 (2.00～6.00) ]. There was significant difference between the two groups(P＜0.001)3. The expression of IL-8 in unbilical cord blood in each groups:The expression of IL-8 were detected in umbilical cord blood by enzyme-linked immunosorbent assay method in 2 groups, including pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The expression, of IL-8 was the higher in pregnant with subclinical chorioamnionitis group (33.43±12.98ng/ml) than those in pregnant without subclinical chorioamnionitis group (20.21±11.47ng/ml) .There was significant difference between the two groups(P＜0.001).4. The expression of IL-8 in maternal blood in each groups:The expression of IL-8 were detected in maternal blood by enzyme-linked immunosorbent assay method in 2 groups, which included the pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of IL-8 was the higheer in pregnant with subclinical chorioamnionitis group[45.14 (25.15～63.21) ng/ml]than those in pregnant without subclinical chorioamnionitis group[24.18 (0.57～65.13) ng/ml].There was significant difference among the two groups(P＜0.001).5. The dependable comparison of IL-8 in placenta and fetal membrane, umbilical cord blood, maternal blood:To analyze the correlation of IL-8 in afterbirth, umbilical cord blood and maternal blood ,we found that the positive correlation was exist in placenta and fetal membrane and umbilical cord blood in expression of IL-8(r=0.296,p＜0.01); The concentration of IL-8 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.304,p＜0.01); The concentration oflL-8 in umbilical cord blood was positively correlated with that of maternal blood (r=0.532,p＜0.01); 6. The ROC curve of IL-86.1 The ROC curve of IL-8 in umbilical cord blood:The critical point of IL-8 in umbilical cord blood is 25.40ng/ml, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve is 0.773. The sensitivity and specificity were 85.3% and 70.1% respectively.6.2 The ROC curve of IL-8 in maternal blood:The critical point of IL-8 in maternal blood is 34.76ng/ml, which is the optimization value to estimate chorioamnionitis. The integral area under the curve is 0.798. The sensitivity and specificity were 79.4% and 79.2% respectively.Conclusion:1. The high expression of the interleukin-8 in maternal blood in subclinical Chorioamnionitis, could be one of an biochemical marker in predicting subclinical Chorioamnionitis and in evaluating the therapeutic effect on subclinical Chorioamnionitis patients.2. The high expression of the interleukin-8 in umbilical cord blood in subclinical Chorioamnionitis, could be one of an biochemical marker in predicting subclinical chorioamnionitis and in evaluating the therapeutic effect on subclinical chorioamnionitis patients.3. It was high expression of the interleukin-8 in placenta and fetal membrane in subclinical Chorioamnionitis that indicates the participation of the morbility of subclinical Chorioamnionitis.4. The high expression of the interleukin-8 in core blood in fetals with subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting and assisting to the early diagnostic of infectious of newborn, and also could be one of an biochemical marker in evaluating the therapeutic effect on infectious of newborn. Objective: To investigate the change of the matrix metalloproteinase-9 in maternal blood, umbilical cord blood, placenta and fetal membrane in subclinical Chorioamnionitis, to analyze its effect on the pathog.enesis of subclinical Chorioamnionitis, to estimate the clinical value of the matrix metalloproteinase-9 anticipation in predicting subclinical Chorioamnionitis.Methods: To Chose 36 cases ofpreterm birth patients in labor , 14 cases of threatened premature labor, and 37 cases full term gravida with spontaneous in labor, 38 cases full term gravida without threatened labor and 18 cases who have the identical gestational age with preterm birth patients . It were enzyme-linked immunosorbentassay, HE staining, immunohistochemical staining that used to observe the change of the matrix metalloproteinase-9 in maternal blood , umbilical cord blood, placenta and fetal membrane in preterm birth patients with subclinical Chorioamnionitis.Results:1. The pathematology results of placenta and fetal membrane in each groups:The pathematology results of placenta and fetal membrane were the same as that of the Chapter I.2. Compared the expression of MMP-9 in placenta and fetal membrane by IHC in each groups:The expression of MMP-9 were detected in 2 groups by immunohistochemistry staining method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group.The median and range of the average staining intensity of MMP-9 was the higher in pregnant with subclinical chorioamnionitis group[6.00 (2.00～8.00) ] than those in pregnant without subclinical chorioamnionitis group[2.00 (2.00～6.00)].There was significant difference between the two groups(P＜0.001).3. The expression of MMP-9 in umbilical cord blood in each groups:The expression of MMP-9 were detected in umbilical cord blood by enzyme-linked immunosorbent assay method in 2 groups, including pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The expression of MMP-9 was the higher in pregnant with subclinical chorioamnionitis group (368.41±110.55ng/ml) than those in pregnant without subclinical chorioamnionitis group (281.67±96.73ng/ml) .There was significant difference between the two groups(P＜0.001).4. The expression of MMP-9 in maternal blood in each groups:The expression of MMP-9 were detected in maternal blood by enzyme-linked immunosorbent assay method in 2 groups, which included pregnant with subclinical chorioamnionitis group and pregnant without subclinical chorioamnionitis group. The median and range of the expression of MMP-9 was the higheer in pregnant with subclinical chorioamnionitis group[458.19 (315.52～522.15) ng/ml]than those in pregnant without subclinical chorioamnionitis group[307.15 (181.29～533.30) ng/ml].There was significant difference among the two groups(P＜0.001).5. The correlation of MMP-9 in placenta and fetal membrane , umbilical cord blood, maternal blood:To compare the correlation of MMP-9 in placenta and fetal membrane, umbilical cord blood, maternal blood ,we found that the concentration of MMP-9 in placenta and fetal membrane was positively correlated with that of umbilical cord blood (r=0.420,p＜0.01); the concentration of MMP-9 in placenta and fetal membrane was positively correlated with that of maternal blood (r=0.412,p＜0.01); the concentration of MMP-9 in umbilical cord blood was positively correlated with that of maternal blood (r=0.561,p＜0.01).6. The ROC curve of MMP-96.1 The ROC curve of MMP-9 in umbilical cord blood:The critical point of MMP-9 in umbilical cord blood is 317.50ng/ml, which is the optimization value to estimate subclinical chorioamnionitis. The area under the curve is 0.720, the sensitivity and specificity were 73.5% and 71.4%,respectively.6.2 The ROC curve of MMP-9 in maternal blood:The critical point of MMP-9 in maternal blood is 360.81ng/ml, which is the optimization value to estimate chorioamnionitis.The area under the curve is 0.823. The sensitivity and specificity were 87.4% and 72.0% respectively.Conclusion:1. The high expression of the matrix metalloproteinase-9 in maternal blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical Chorioamnionitis and in evaluating the therapeutic effect on subclinical Chorioamnionitis patients.2. The high expression of the matrix metalloproteinase-9 in umbilical cord blood in subclinical Chorioamnionitis, which could be one of an biochemical marker in predicting subclinical chorioamnionitis and in evaluating the therapeutic effect on subclinical chorioamnionitis patients.3. It was high expression of the matrix metatloproteinase-9 in placenta and fetal membrane in subclinical Chorioamnionitis. which indicates that it is the important pathogenetic reason for subclinical Chorioamnionitis.更多还原