【作者】 孙逸；

【导师】 俞惠民；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 目的：早产儿脑白质损伤(white matter damage, WMD)被认为是早产儿最主要也是最重要的脑损伤。WMD的关键性靶细胞是少突胶质前体细胞。最近有学说指出WMD的发病机制中存在少突胶质前体细胞分化障碍。流行病学和动物实验研究均表明宫内感染是早产儿WMD发生的主要因素之一。神经免疫应答反应被认为在宫内感染和早产儿WMD中起重要的介导作用。但具体的分子机制目前还不明确。Toll样蛋白的研究是近年来炎症信号传导领域取得的最重要进展。Toll样受体(Toll-like receptor, TLR)激活后通过诱导一系列炎性相关因子的表达调控机体固有性免疫应答,并最终激活适应性免疫应答。研究指出TLR-4信号通路参与缺血性脑梗塞、脑挫伤、帕金森氏病、蛛网膜下腔出血、多发性硬化、缺氧性脑损伤等一系列免疫应答相关性脑损伤的发生。因此有学者提出：神经免疫应答过程中小胶质细胞TLR-4信号的表达启动在脑内防御反应及脑损伤的平衡机制中起重要作用。最新的研究还指出TLR-4信号能直接激活经典的notch1信号通路分子。而Notch1信号通路通过其下游目的基因Hes (Hairy enhancer of split)基因家族(如Hes1、Hes5等)对少突胶质细胞分化起到重要的负性调控作用。目前国内外对于TLR-4信号在宫内感染致WMD发生中作用的研究不多,且多集中在离体水平上。过去几年间,我们实验室通过子宫颈内接种大肠杆菌的方式成功建立了宫内感染致新生大鼠WMD的动物模型。基于这一成熟模型,本研究观察宫内感染后仔鼠脑内TLR-4信号分子表达。并通过检测相关下游细胞因子、少突胶质细胞分化负性调控因子,进一步探讨TLR-4信号在宫内感染致WMD中的作用机制。该研究为临床治疗早产儿WMD提供丰富的动物实验信息及依据,为今后相关领域深入研究奠定整体动物实验基础,并为进一步研究早产儿WMD的防治提供新的靶点。方法：1.实验分组：将孕鼠随机分为对照组、宫内感染组。宫内感染组孕龄15天的孕鼠宫颈左右两侧各注射大肠杆菌稀释液0.2 ml,对照组同孕龄孕鼠注入等量生理盐水。2.标本采集：选择17、19、21天胎龄和新生1、3、7、14日龄为观察时点,剖腹取出胎鼠或者随机选取各组新生仔鼠断头处死,取出脑组织置于10%中性甲醛或液氮速冻后-80℃超低温冰箱中冷冻保存。3.实验方法：测定各时点仔鼠体重、脑重。应用苏木素-伊红(hematoxylin eosin, HE)染色观察各组新生1、3、7、14日龄大鼠脑室周围白质组织病理变化特点。Western blot检测各时点仔鼠脑组织髓鞘碱性蛋白(myelin basic protein, MBP)、TLR-4和核因子kappa B p65 (nuclear factor-kappa B p65, NF-κB p65)的蛋白表达水平,荧光定量RT-PCR检测各时点仔鼠脑内TLR-4、NF-κB p65、细胞因子肿瘤坏死因子(tumor necrosis factor, TNF)-α.白细胞介素(interieukin, IL)-1β、Notch1、Hes5 mRNA的表达。结果：1.44只孕鼠活动、进食均正常,无1例发生死亡、早产。仔鼠情况：对照组获取新生仔鼠66只,17、19、21天胎龄仔鼠56、55、58只,死产2只,死胎7只；宫内感染组获取新生仔鼠70只,17天、19天、21天胎龄仔鼠57、58、56只,死产5只,死胎8只。宫内大肠杆菌感染的孕鼠子宫壁及胎盘内血管充血、水肿,并见大量中性粒细胞浸润。对照组的孕鼠子宫及病理检测均未见明显的炎症反应。2.宫内感染后,各时点胎鼠及1日龄仔鼠体重、脑重下降.3.7、14日龄仔鼠脑室周围白质部位HE染色可见：对照组脑白质组织结构正常,染色清晰；宫内感染组脑白质染色淡,结构稀疏呈筛网状改变。4.对照组14日龄仔鼠脑组织出现MBP表达,宫内感染组14日龄仔鼠脑组织MBP表达减少甚至消失。5.宫内感染后TLR-4及其下游信号分子NF-κB p65表达在17日胎龄及1、3、7日龄仔鼠脑内同步化上调。6.宫内感染后19、21天胎龄仔鼠脑内TNF-α表达上调。7.宫内感染后Notchl及其下游信号分子Hes5表达在19日胎龄及1、3、7日龄仔鼠脑内同步化上调。结论：1.宫内感染可使胎鼠体重、脑重下降。2.TLR-4信号可能参与宫内感染致WMD的发生。3.TLR-4信号可能通过调控细胞因子的的表达部分介导宫内感染后WMD的发生。4.TLR-4信号在宫内感染致脑白质病变的发生中的作用可能部分源于激活的Notchl信号阻碍少突胶质细胞前体分化。更多还原

【Abstract】 Objective:White mater damage (WMD) in the premature babies is considered as the most common and important form of injury to the preterm brain. Preoligodendrocyte (preOLs) is the key target cell of WMD. Recently, arrest of preOLs differentiation was reported to be involved in WMD. The epidemiologic evidence and study on animal models supported the view that one of the most important factors that associated with WMD appeared to be intrauterine infection. Neural immune response has long been considered to play an important role in the WMD induced by intrauterine infection. But the exact molecular mechanism is still unclear.The identification and the study of Toll-like protein is one of the most advances in the field of inflammatory signal transduction. Activation of TLRs recruits downstream signaling proteins, results in transcription of genes encoding inflammation-associated molecules. TLRs control innate immune response and finally initiate adaptive immune response. A series of studies pointed out that TLR-4 signal pathway was involved in many brain injury associated with immune response such as ischemic stroke, Parkinson disease, multiple sclerosis, and so on. Thus, some researchers proposed a key role of the activation of TLR-4 signal pathway in the balancing mechanism of the hurt and the defense in brain upon stimulation. What is more, some studies confirmed lately that activation of TLR-4 signal pathway can directly induce expression of canonical notch target genes. Notch1 signal pathway disturbs the differentiation of oligodendrocytes by inducing the expression of the downstream target genes such as Hairy enhancer of split gene 5 (Hes5).Now, few researches focused the effect of TLR-4 signal on the WMD induced by intrauterine infection especially in vivo level. In past several years, our laboratory has successfully established an animal model of neonatal rat WMD induced by intrauterine infection after endocervical inoculation with Escherichia coli (E. coli) suspension. On the basis of this stable animal model, we observed the expression of TLR-4 signal molecules in the developing brain (including fetal rats and neonatal rats) after intrauterine infection in our present study. To make further efforts to investigate the potential mechanism by which TLR-4 signal contributes to the development of the WMD after intrauterine infection, we detected the expression of TLR-4 associated downstream cytokines and negative regulatory factors during preOLs maturation. This study may offer lots of experimental information and evidence on the clinical diagnosis and treatment of preterm WMD, may constitute a solid experimental basis in "in vivo" level for further study in correlative field, and may provide new targets for the future treatment and prevention of preterm WMD.Methods:1. Group:All rats were randomly divided into control group and intrauterine infection group.In the intrauterine infection group, pregnant rats at 15 days of gestation were inoculated endocervically with 0.4 mL of E.coli suspension. While in the control group, the rats were injected endocervically with 0.4 mL of sterile saline solution instead.2. Sample collection:The developing rats(including fetuses delivered by cesarean section and neonates) were sacrificed by decapitation at embryonic day 17(E17), E19, E21 and postnatal day 1 (P1),P3 and P7, P14, and the brains were fixed in neutral formaldehyde or stored at-80℃freezer.3. Experimental methods:The body weight and brain weight of all the developing rat in both groups were recorded. Hematoxylin-eosin (HE) staining determined the cerebral WMD of neonatal rats at P1, P3, P7 and P14. Western blot analysis were used for evaluation of myelin basic protein(MBP), TLR-4 and nuclear factor-kappa B p65 (NF-κB p65) protein levels in developing rat brains at E17, E19, E21, P1, P3, P7, P14. Real-time quantitative RT-PCR was used to analyze TLR-4, NF-κB p65, tumor necrosis factor (TNF)-a, interleukin (IL)-1β, as well as Notch 1, Hes5 mRNA expression in developing rat brains at E17, E19, E21, P1, P3 and P7.Results1. Forty-four dams had normal food intake and activity, and none died or delivered immature fetuses after inoculation. In the control group,66 neonatal rats,56 fetal rats of E17,55 fetal rats of E19,58 fetal rats of E21 were collected. There were 7 stillborn fetuses,2 stillborn neonates in the control group. In the intrauterine infection group,70 neonatal rats,57 fetal rats of E17,58 fetal rats of E19,56 fetal rats of E21 were collected. There were 7 stillborn fetuses,2 stillborn neonates in the intrauterine infection group. Inflammation of the uterus and the placenta was induced after intrauterine E. coli inoculation, but all cases of the control group had no histologic evidence of intrauterine infection.2. After intrauterine infection, the body weight and brain weight of E17, E19, E21 fetal rats and P1 neonatal rats significantly decreased.3. HE staining procedures revealed clear staining and normal structure of periventricular white matter from P7, P14 rats of the control group. Weak staining and focal rarefaction of periventricular white matter were shown in P7, P14 rats of the the intrauterine infection group.4. The expression of cerebral MBP was detected in P14 rats of the control group, while the expression of MBP in the brain of P14 rats decreased or disappeared after intrauterine infection.5. The expressions of cerebral TLR-4 and downstream signal molecule NF-κB p65 increased significantly in phase after intrauterine infection at E17, P1, P3, P7.6. The expression of cerebral TNF-a mRNA was up-regulated at P19, P21 after intrauterine infection.7. The expressions of cerebral Notchl and downstream signal molecule Hes5 increased significantly in phase after intrauterine infection at E19, P1, P3, P7.Conclusion:1. Intrauterine infection decreased the body weight, the brain weight of fetal rats.2. TLR-4 signal might play a certain role in the development of the WMD induced by intrauterine infection.3. TLR-4 signal might contribute to the development of the WMD after intrauterine infection partly by regulating the expression of certain cytokines such as TNF-a.4. TLR-4 signal might contribute to the development of the WMD after intrauterine infection partly by activating Notchl signal that arrested the differentiation of preoligodendrocytes.更多还原