【作者】 刘艳；

【导师】 罗小平；

【作者基本信息】 华中科技大学 ， 儿科学， 2009， 博士

【摘要】 目的近年来大量的临床和动物实验研究均证实，宫内感染、炎症反应是导致新生儿脑损伤的重要因素。宫内感染时，革兰阴性细菌细胞壁上的一种大分子结构成分脂多糖(LPS)是重要的致病因素之一。LPS可透过胎盘诱导细胞因子前体的增加，并使胎儿血液循环中单核细胞产生IL-1β和TNF-α等，从而增加了血脑屏障的通透性。LPS和细胞因子得以通过血脑屏障进入胎儿的中枢神经系统，引起LPS介导的小胶质细胞激活，通过一系列的炎症反应诱导神经细胞的凋亡与氧化损伤，最终导致脑损伤的发生。本课题试图建立宫内感染致脑损伤动物模型，并检测胎鼠及新生大鼠脑中TLR4、核因子-kB及细胞因子TNF-αmRNA在这一损伤中的表达变化，探讨其在LPS致脑损伤发生中的作用。方法本实验中取孕龄17d、18d(足月为22．5d)的SD大鼠，每次350μg/kg，连续2d腹腔注射，构建宫内感染大鼠模型(LPS组)；对照组孕鼠腹腔注射同剂量的生理盐水(NS组)。观察胎盘和脑组织炎症细胞侵润和病理改变。分别留取孕20、21d及生后1、3、7、14d(P1、P3、P7、P14)的新生大鼠脑标本，采用Real-timePCR方法检测细胞因子TNF-α、TLR4、核因子-kB在mRNA水平的表达，同时通过Western blot方法检测蛋白水平的表达情况。结果实验中各组孕鼠经腹腔注射后活动、进食均正常。LPS组孕鼠有1例发生死亡，4例术后出现阴道流血并流产，1例有死产。LPS组新生鼠平均出生体重较NS组明显减低(p＜0．05)。HE染色可见LPS组孕鼠胎盘组织间质显著增生，毛细血管腔变窄，并可见明显炎性细胞浸润。LPS组新生大鼠脑组织HE染色可见细胞水肿，组织疏松，细胞数减少。蛋白表达结果显示LPS组TLR4明显升高，较NS组有显著性差异，F=71．148，p=0．001；LPS组NF-kB较NS组有显著性差异，F=47．844，p=0．002；LLPS组TNF-a较NS组有显著性差异，F=16．863，p=0．015。LPS组以上三种蛋白测量值的平均值均大于NS组。用Realtime PCR进行定量分析发现LPS组的TLR4、NF-kB、TNF-αmRNA在G20、G21、P1、P3、P7的表达比NS组明显升高，有显著性差异(p＜0．05)，而在P14两组无显著差异(p＞0．05)。结论宫内LPS感染可导致胎盘组织以及胎儿的炎性反应，进而激活脑内的免疫系统，使小胶质细胞活化。LPS介导的TLR4信号途径被激活，诱导炎性因子大量释放，最终导致脑损伤的发生。注射LPS后仔鼠脑中炎性因子表达明显增强，而P14新生大鼠脑内炎性因子的表达与对照组已无明显差异，说明宫内感染所致的早期炎症级联反应在脑损伤的发病机制中起着至关重要的作用。目的近年来大量的临床和动物实验研究均证实，宫内感染、炎症反应与脑损伤密切相关。宫内感染后脑内小胶质细胞激活，LPS信号途径活化，TLR4在LPS信号转导途径中起着十分重要的作用，最终导致转录因子NF-kB的激活，导致炎性细胞因子的合成和释放，脑中炎症级联反应，导致前体少突胶质细胞凋亡，星形胶质细胞增生，髓鞘化减低，从而产生脑白质损伤。DHA属ω-3系列长链多不饱和脂肪酸，是大脑细胞膜的重要构成成分，具有抗氧化功能。从确认怀孕第一天起即给予不同剂量的DHA灌胃，探讨DHA干预后对宫内感染致脑损伤的影响。方法本实验中取孕龄17d、18d(足月为22．5d)的SD大鼠，每次350μg/kg，连续2d腹腔注射，构建宫内感染大鼠模型，并分成三组：B为对照组，自由饮水和进食；实验C组(DHA1组)每日半粒鱼油(DHA 65mg)灌胃；实验D组(DHA2组)每日一粒鱼油(DHA 130mg)灌胃，其余饮食均同对照组。分别留取孕20、21d及生后1、3、7、14日龄(P1、P3、P7、P14)的新生鼠脑标本，通过Real-timePCR方法检测细胞因子TNF-α、TLR4、核因子-kB在mRNA水平的表达情况，同时通过Western方法检测蛋白表达情况来评价胎鼠及新生鼠脑损伤情况。结果实验中各组孕鼠经腹腔注射后活动、进食均正常。LPS组孕鼠有3例术后流产，DHA1干预组一例流产，DHA2干预组无一例流产，三组均无孕鼠死亡。LPS组新生鼠平均出生体重较DHA干预组组明显减低(P＜0．05)。孕鼠胎盘组织HE染色可见LPS组炎性浸润明显，而DHA干预组炎性浸润减轻，血供较LPS组丰富。LPS组脑组织HE染色见细胞水肿，组织疏松，细胞数减少。DHA干预组脑细胞水肿减轻，细胞数减轻不明显。蛋白表达结果显示不同剂量的DHA干预组TLR4表达较LPS组减少(F=38．944，p＜0．001)，不同剂量的DHA干预组NF-kB表达较LPS组减少(F=18．997，p=0．003)，不同剂量的DHA干预组TNF-α表达较LPS组减少(F=22．556，p=0．002)。Real-time PCR方法检测到DHA1、DHA2组的TLR4、NF-kB、TNF-αmRNA整体水平较LPS组下降，有统计学意义(p＜0．05)，但每个时间点各有差异。结论宫内LPS感染可导致胎盘组织以及胎儿的炎性反应，激活LPS介导的TLR4信号途径，导致脑损伤的发生。DHA是鱼油的主要成分，也是中枢神经系统必不可少的多不饱和脂肪酸，在神经发育和功能方面有着重要作。在孕期补充DHA后，宫内感染的胎鼠及新生鼠脑细胞水肿减轻，细胞数增加，TLR4、NF-kB及TNF-αmRNA及蛋白表达都明显下降，说明DHA在针对LPS所致的宫内感染中有抗炎效应，能调节脑内致炎细胞因子的表达。更多还原

【Abstract】 Objective In the resent years,epidemiologic and experimental findings showed there wasa close association between proinflammatory cytokines production and intrauterineinfection-induced brain damage.LPS,as a giant molecule on the cell wall of Gram negativebacteria,is one of the important etiological factors during intrauterine infection.LPS cancross through the placenta and induce cytokine precurosor increasing.Monocyte of fetalcirculation product IL- 1β,TNF-α,thus the permeability of the blood brain barrier increase.LPS and cytokine can pass the blood brain barrier and get into fetal central nervous system,evoking LPS mediated gitter cell activation.These inflammatory responses induce nervecell apoptosis and oxidative damage,and finally result in neonatal brain white matterdamage.The present study will establish the intrauterine infection rat model and probe intothe relationship between neonatal brain damage and inflammatory response throughidentifying the expression of TLR4、NF-κB and TNF-αmRNA in fetal and neonatal brain.Methods In the present study,Sprague-Dawley (SD) rats (n=20) at day 17 and 18 ofgestation underwent treatment for two days.We set up the intrauterine infection rat model(LPS-treated group) through intraperitoneal LPS administration (350μg/kg.dX2),while thecontrol group was administrated of the same volume of pyrogen-free saline (NS-treatedgroup).Brain tissues were collected from the fetal rat at pregnant day 20,21 and neonatalrat at postnatal day 1 (P 1),P3,P7 and P 14.Real-time quantitative polymerase chain reaction(RT-PCR) analysis was used to test mRNA expression for TLR4,NF-κB and TNF-αandWestern blotting was used to evaluate the TLR4,NF-κB and TNF-αexpression in thebrain tissues.Results Pregnant rats of each group had normal activity and feeding after intraperitonealinjection.One pregnant rat was dead,four pregnant rats were aborted and one had still birthin the LPS-treated group.The mean birth weight of the rats in the LPS- treated group was significant lower than the control group (p＜0.05).Placental hematoxylin-eosin staining showed remarkable interstitial hyperplasia,narrow capillary andinflammatory infiltration.Brain hematoxylin-eosin staining showed cellular edema,tissueraritas and cell population decreased.Protein express showed TLR4 in LPS-treated groupwas significantly increased compared to that of the control group (F=71.148,p=0.001).NF-κB expression in LPS-treated group was significantly higher than the control group(F=47.844,p=0.002).TNF-αin the LPS-treated group was significantly increased than thatof the control group (F=16.863,p=0.015),and the mean values of the three proteins in LPS-treated group were much higher when compared to the control group.The expression ofTLR4,NF-κB,TNF-αmRNA in LPS-treated group were significantly increased comparedto that of the control group in G20,G21,P1,P3,P7 rat brain with real-time PCRmeasurements (p＜0.05).There was no significant difference on TLR4,NF-κB and TNF-αmRNA levels between the LPS-treated group and the NS-treated group at the P14neonatal rat brain (p＞0.05).Conclusions Intrauterine LPS exposure could induce an inflammatory response of theplacenta and the fetus,then activate the intracephalic immune system.Activation of gittercell and LPS mediated TLR4 signal pathway could induce the releasing of inflammatoryfactor greatly,and eventually result in brain injury.After intraperitoneal injection of LPS,the express of inflammatory factors were remarkably increased in the fetus and neonatalbrain in P1,P3 and P7.But there was no significant difference in the expression ofproinflammatory factors in the neonatal brain in P 14 between the LPS-treated group and theNS-treated group.This suggested that the early inflammatory cascade response played animportant role in the pathogenesis of neonatal brain damage. Objective In the resent years,clinical and animal experimental findings showed therewas a close association between inflammatory reaction,intrauterine infection andneonatal brain damage.Gitter cells in the brain were activated and then LPS signal pathwaywas activated after intrauterine infection.TLR4 played a very important role in the LPSsignal transduction pathway.It induced nuclear factor (NF-κB) activation andproinflammatory cytokines synthesis and release.Inflammation cascade reaction in thebrain resulted in oligodendrocyte progenitor cell apoptosis,horizontal cell proliferation andhypomyelination.These inflammatory response finally resulted in neonatal white matterdamage.DHA is one of theω-3 polyunsaturated fatty acid.It is one of the importantcomponents of cerebral cellular membrane,which has antioxydation function.Intragastricadministration of fish oil to the pregnant rats during the whole pregnancy in this currentstudy.We will probe the effect of supplying DHA on the neonatal rat brain of intrauterineLPS exposure duration of pregnancy.Methods In the current study,Sprague-Dawley (SD) rats (n=20) at day 17 and 18 ofgestation underwent operation continue two days.We set up the intrauterine infection ratmodel (LPS-treated group) through intraperitoneal LPS administration (350μg/kg/d).Pregnant Sprague-Dawley rat were divided into three groups:control group (LPS-treatedgroup,freedom drinking water and eating);DHA1-treated group (intragastricadministration of half pill of fish oil during the whole pregnancy);DHA2-treated group(intragastric administration of one pill of fish oil during the whole pregnancy).Braintissues were collected from the fetal rat at pregnant day 20,21 and neonatal rat at postnatalday 1 (P1),P3,P7 and P14.Real-time quantitative polymerase chain reaction (RT-PCR)analysis was used to test mRNA expression for TLR4,NF-κB and TNF-αand Westernblotting was used to evaluate of TLR4,NF-κB and TNF-αexpression in brain tissues.Results Pregnant rats of each group had normal activity and feeding after intraperitonealinjection.Three pregnant rats were aborted in the LPS-treated group.One pregnant rat was aborted in the low dose DHA-treated group.There was no pregnant rat aborted in the largedose DHA-treated group.There was no pregnant rat died in the three groups.The meanbirth weight of the LPS-treated group neonatal rats was significant lower than the DHA-treated group (p＜0.05).Placental hematoxylin-eosin staining showed obvious inflammatoryresponse in the LPS-treated group.Inflammatory infiltration of the placental in the DHA-treated group was decreased.Brain hematoxylin-eosin staining showed cellular edema,tissue raritas and cell population decreased in the LPS-treated group.Brain cellular edemawas relieved and cell population was not obviously decreased in the DHA-treated group.Protein express showed TLR4 in DHA-treated group was significantly increasing than inLPS-treated group (F=38.944,p＜0.001).NF-κB in DHA-treated group was significantlyincreasing than in LPS-treated group (F=18.997,p=0.003).TNF-αin DHA-treated groupwas significantly increasing than in LPS-treated group (F=22.556,p=0.002).Real-timePCR showed general average of TLR4、NF-κB and TNF-αmRNA in DHA1 and DHA2treated group were significantly decreasing than in LPS-treated group.But there wasdistinction at different time point.Conclusions Intrauterine LPS infection could induce an inflammatory response ofplacenta and fetus,then activate LPS mediated TLR4 signal pathway.The inflammatoryresponse finally resulted in brain injury.DHA was one of the main components of fish oiland indispensable polyunsaturated fatty acid in the central nervous system.It played animportant role in neural development and function.Fetus and neonatal brain cellular edemawas decreased and cell population was increased after supplement of DHA during thewhole pregnancy.Protein of TLR4,NF-κB,TNF-αand TLR4,NF-κB,TNF-αmRNA weredecreased significantly.We proposed that DHA is highly anti-inflammatory by targetingLPS mediated intrauterine infection.DHA could modulate the expression ofproinflammatory cytokines in the brain.更多还原