宫内炎症与早产儿临床结局及LPS诱导脐血免疫细胞TLR信号通路基因表达谱特征的研究

发布时间：2018-06-10 11:28

本文选题：宫内炎症 + 母体炎症反应　；参考：《复旦大学》2012年博士论文

【摘要】：胎儿／新生儿期不仅是人一生中最易感染的时期,而且感染死亡率高。超过50%的新生儿死亡与宫内／宫外感染有关。27%的新生儿死亡是由早产所致,而造成早产的一个重要因素是宫内感染。另外,26%的新生儿死亡原因为败血症和肺炎。除了引起早产外,宫内感染还可能影响早产儿的近、远期临床结局。胎儿／新生儿对病原菌的易感性是由自身免疫特点决定。本课题通过较大样本的队列,前瞻性研究了宫内感染／炎症与早产新生儿临床结局的关系。并通过比较分析了新生儿脐血免疫细胞TLR信号通路基因表达谱特征,初步探讨了胎儿／新生儿感染性疾病易发的分子机制,以期进一步丰富对胎儿／新生儿感染这一重要临床问题的认识。第一部分宫内炎症与早产儿临床结局的研究目的研究宫内炎症与早产的关系以及对新生儿呼吸系统、脑损伤等临床结局的影响,为临床的有效干预提供依据。方法2008年1月至2010年10月在在上海交通大学附属国际和平妇幼保健院分娩、孕周34周的孕产妇,及其所分娩的216名单胎早产儿纳入本研究。收集产前、产时以及新生儿临床资料并输入数据库。对于转出新生儿,随访并收集数据。根据胎盘病理,将宫内炎症分为母体炎症反应(Maternal inflammation response,MIR)和胎儿炎症反应(Fetal inflammation response, FIR)。其中95例早产儿完成床旁头颅超声筛查；76例产妇行宫腔支原体和细菌培养；36例早产儿于纠正胎龄1-3月完成头颅MRI检查；25名早产儿于纠正胎龄6月进行听觉脑干诱发电位检查,以及于纠正胎龄12个月采用贝利发育量表进行神经发育评估。结果(1)胎盘病理：104/216例(48.1%)胎盘病理检查发现宫内炎症反应,其中53例(51.0%)仅为母体炎症反应,51例(49.0%)炎症反应同时累及母体和胎儿。本组资料未见仅为胎儿炎症的病理结果。根据炎症反应病理结果将早产儿分为母体和胎儿炎症双阴性组(MIR-FIR-组)、母体炎症阳性而胎儿炎症阴性组(MIR+FIR-组)以及母体和胎儿炎症双阳性组(MIR+FIFR+组)。(2)早产与宫内炎症的关系：依据胎龄将216名早产儿分为27-29周、30-32周以及33周三组。胎龄27-29周和30-32周两组MIR+(不论是否累及胎儿)发生率分别50.0%和54.8%,显著高于胎龄≥33周组35.4%的发生率(p0.05)。胎龄27-29周和30-32周两组MIR+FIFR+发生率分别为27.8%和28.7%均显著高于胎龄≥33周组12.1%的发生率(p0.05)。而三组MIR+FIR-发生率则无显著性差异。(3)宫腔微生物培养结果：支原体阳性率为22.4%,细菌阳性率为18.4%,各组间支原体和细菌培养阳性率无差异。(4)宫内炎症与新生儿脑损伤的关系：95名早产儿完成头颅超声检查,纳入分析。尽管三组间脑室内出血发生率总体无差异,但MIR+FIR+组脑室内≥2度脑室内出血发生率显著高于其他两组。logistic多元回归分析发现MIR+FIR+显著增加早产儿2度及以上脑室内出血风险,OR=4.08(95%CI,1.259-13.24)。(5)宫内炎症与新生儿呼吸窘迫综合症的关系：MIR+FIR-组和MIR+FIR+组RDS发生率显著低于MIR-FIR-组,且早产儿需机械通气率也显著降低。logist多元回归分析发现MR+FIR+显著降低RDS的风险,OR=0.08(95%CI,0.010-0.661)。(6)宫内炎症与新生儿败血症的关系：MIR+FIR+组、MIR+FIR-组和MIR-FIR-组早产儿早发型败血症发生率分别为15.7%、11.3%和7.1%。虽然宫内炎症暴露增加早发型败血症的趋势明显,但无统计学差异。(7)部分随访结果：对25名早产儿于纠正胎龄6月进行听觉脑干诱发电位检查,以及于纠正胎龄12个月采用贝利发育量表进行神经发育评估,根据MIR(不论是否累及胎儿)将早产儿分为两组。宫内炎症对听觉功能、智力发展指数和运动发展指数无影响。结论(1)胎盘、胎膜和脐带病理是判断宫内炎症的重要依据。母体炎症反应与胎儿炎症反应是既密切相关又明显不同的病理现象；(2)低胎龄早产时宫内炎症更严重；伴有胎膜早破时,宫内炎症与胎龄呈负相关；(3)宫内炎症影响新生儿期临床结局。宫内炎症累及胎儿时显著增加2度及以上脑室内出血的风险；宫内炎症累及胎儿时显著降低新生儿RDS发生风险；宫内炎症有增加早产儿早发型败血症的趋势；第二部分LPS诱导脐血免疫细胞TLR信号通路基因表达特征的研究目的通过比较新生儿脐血与成人外周血免疫细胞经LPS刺激后TLR信号通路基因表达谱的差异性,探讨新生儿抗感染固有免疫应答的特点和易于感染的分子机制。方法收集10份健康足月儿脐血和10份成人外周血,进行全血细胞培养。分别于脂多糖(lipopolysaccharide, LPS)诱导刺激后0、2、4、6、8和24小时,分离收集单个核细胞,抽提RNA。采用SuperArray公司(USA)Oligo GEArray(?)人Toll样受体(TLR)信号通路功能基因芯片,检测分析脐血TLR信号通路基因表达谱的时序变化特点及与成人的差异,并应用PCR array对结果进行验证。结果基因表达谱芯片结果显示,新生儿较成人差异性表达(≥2倍)的基因随时间点不同而变化。LPS诱导后8小时,新生儿较成人差异性表达的基因个数达到最大,共有79个基因,其中上调40个基因,下调基因39个。时序特征分析提示LPS诱导脐血免疫细胞TLR信号通路基因表达于诱导后2小时达高峰,但持续时间较短,而成人则于诱导后6小时达高峰,持续时间较长。脐血部分基因在所有时间点或大部分(≥4个点)时间点上调或下调变化趋势较为一致,如表达上调的基因有RP105、Tollip、GPC1、HRAS、HSPA4、FADD、IKKα、NFKBIL1、SAPK3、JNK2和JNK1,而表达下调基因有TLR1、TLR2、CD14、MyD88、HSPA1A、MAL、 IRAK3和RELB等。新生儿TLR信号通路中一些重要的负性调控基因表达较成人高,而促炎因子的表达普遍低于成人。结论(1)LPS诱导的脐血TLR信号通路基因表达水平及时序变化与成人存在明显差异；(2)新生儿TLR信号通路的基因表达特征以负性调控占优势,可能是其抗感染免疫应答不足的重要机制之一。
[Abstract]:Fetal / neonatal period is not only the most susceptible period of human life, but also the mortality rate is high. More than 50% of newborn deaths associated with intrauterine / intrauterine infection related to.27% are caused by premature birth, and intrauterine infection is an important factor causing premature birth. In addition, 26% of the causes of neonatal death are septicaemia and pneumonia. Intrauterine infection may also affect the near and forward clinical outcome of preterm infants. The susceptibility of the fetus / newborn to the pathogenic bacteria is determined by the autoimmune characteristics. This subject has prospectively studied the relationship between intrauterine infection / inflammation and the clinical outcome of premature neonates through a large sample of samples. In order to further enrich the understanding of the important clinical problems of fetal / neonatal infection, the molecular mechanism of the TLR signaling pathway in fetal umbilical cord blood immune cells is preliminarily discussed.
Part one: intrauterine inflammation and clinical outcome of premature infants
Objective to study the relationship between intrauterine inflammation and premature delivery, and the effect on neonatal respiratory system and brain damage, so as to provide evidence for effective intervention in clinic.
Methods from January 2008 to October 2010, the pregnant and parturient women who delivered at the International Peace Maternity and child health care hospital, affiliated to Shanghai Jiao Tong University, 34 weeks of pregnancy, and 216 of the birth preterm infants were included in this study. The data of prenatal, intrapartum and neonatal clinical data were collected and entered into the database. The intrauterine inflammation was divided into Maternal inflammation response (MIR) and fetal inflammatory response (Fetal inflammation response, FIR). 95 preterm infants were screened by bedside cranial ultrasound screening; 76 cases of parturients were infected with Mycoplasma and bacteria; 36 premature infants completed 1-3 months' head MRI examination; 25 The premature infants were examined by auditory brainstem evoked potential in June and corrected for 12 months of gestational age. The Bailey developmental scale was used to assess neural development.
Results (1) placental pathology: 104/216 (48.1%) placental pathological examination found intrauterine inflammatory reaction, of which 53 cases (51%) were only maternal inflammatory reaction, 51 (49%) inflammatory reaction involved both mother and fetus. The data of this group were not only the pathological results of fetal inflammation. According to the pathological results of the inflammatory reaction, the preterm infants were divided into maternal and fetal inflammation. MIR-FIR- group (group MIR-FIR-), maternal inflammation positive and fetal inflammation negative group (group MIR+FIR-) and maternal and fetal inflammation double positive group (group MIR+FIFR+). (2) the relationship between premature birth and intrauterine inflammation: according to gestational age, 216 preterm infants were divided into 27-29 weeks, 30-32 weeks and 33 Wednesday groups. 27-29 and 30-32 weeks of fetal age, two groups, whether or not involved. The incidence of fetal) was 50% and 54.8% respectively, which was significantly higher than the incidence of 35.4% in the 33 weeks group (P0.05). The incidence of MIR+FIFR+ in two groups of two groups at the 27-29 and 30-32 weeks of gestational age was significantly higher than that of 12.1% in the gestational age group (33 weeks). The incidence of MIR+FIR- in the three group was no significant difference. (3) the results of microbiological culture in the uterine cavity were: The positive rate of Mycoplasma was 22.4% and the positive rate of bacteria was 18.4%. There was no difference in the positive rate of Mycoplasma and bacteria in each group. (4) the relationship between intrauterine inflammation and neonatal brain injury: 95 preterm infants completed the skull ultrasound examination and included the analysis. Although there was no difference in the incidence of intraventricular hemorrhage in the three groups, the cerebral ventricles of the MIR+FIR+ group were more than 2 degrees in the ventricles of the brain. The incidence of bleeding was significantly higher than the other two groups of.Logistic multivariate regression analysis found that MIR+FIR+ significantly increased the risk of intraventricular hemorrhage of 2 degrees and above in preterm infants, OR=4.08 (95%CI, 1.259-13.24). (5) the relationship between intrauterine inflammation and neonatal respiratory distress syndrome: the incidence of RDS in MIR+FIR- and MIR+ FIR+ groups was significantly lower than in the MIR-FIR- group, and the need for premature infants The rate of mechanical ventilation was also significantly reduced by.Logist multivariate regression analysis found that MR+FIR+ significantly reduced the risk of RDS, OR=0.08 (95%CI, 0.010-0.661). (6) the relationship between intrauterine inflammation and neonatal septicemia: the incidence of early onset sepsis in premature infants in group MIR+FIR+, MIR+FIR- and MIR-FIR- was 15.7%, 11.3% and 7.1%. although increased intrauterine inflammation increased The trend of early onset sepsis was obvious, but there was no statistical difference. (7) part of the follow-up results: 25 preterm infants were examined by auditory brainstem evoked potential in June, and the Bailey development scale was used for the assessment of neurodevelopment in 12 months of gestational age. Preterm infants were divided into two groups according to whether or not they were involved in the fetus. Inflammation had no effect on auditory function, intelligence development index and motor development index.
Conclusions (1) the pathology of placenta, fetal membrane and umbilical cord is an important basis for judging intrauterine inflammation. Maternal inflammatory reaction and fetal inflammation are closely related and distinct pathological phenomena; (2) intrauterine inflammation is more severe in premature birth of low fetal age; intrauterine inflammation is negatively correlated with fetal age, and (3) intrauterine inflammation affects newborns The risk of intrauterine intrauterine bleeding increased by 2 degrees and above, with intrauterine inflammation involving the fetus; intrauterine inflammation significantly reduced the risk of RDS in the newborn; intrauterine inflammation increased the trend of early onset septicemia in preterm infants;
The second part is about the gene expression characteristics of TLR signaling pathway induced by LPS in umbilical cord blood immune cells.
Objective to compare the difference of the gene expression profiles of TLR signaling pathway in neonatal umbilical blood and adult peripheral blood immune cells after LPS stimulation, and to explore the characteristics of neonatal anti infection inherent immune response and the molecular mechanism that is easy to infect.
Methods 10 healthy full moon umbilical cord blood and 10 adult peripheral blood were collected for whole blood cell culture. 0,2,4,6,8 and 24 hours after induced stimulation of lipopolysaccharide (LPS) were separately collected and collected to collect mononuclear cells, and RNA. was extracted from SuperArray company (USA) Oligo GEArray (?) Toll like receptor (TLR) signal pathway function gene chip. The temporal variation characteristics of umbilical cord blood TLR signaling pathway gene expression and its difference from adults were detected and analyzed, and the results were verified by PCR array.
The results of gene expression chip showed that the genes of differential expression (2 times greater than 2 times) of the newborns were different with time, and 8 hours after the induction of.LPS induction. The number of different genes expressed in the newborns reached the maximum, with a total of 79 genes, including 40 genes up and 39 down regulated genes. The sequence characteristic analysis suggested that LPS induced cord blood. The expression of TLR signaling pathway in the immune cells reached the peak at 2 hours after induction, but the duration of the gene was shorter, while adults reached the peak at 6 hours after induction, and lasted a long time. The genes in the umbilical cord blood were up to be up or down at all time points or at most (4 points) time points, such as RP105, Tollip, in the up regulated genes. GPC1, HRAS, HSPA4, FADD, IKK alpha, NFKBIL1, SAPK3, JNK2 and JNK1. Some of the important negative regulatory genes in the signaling pathway are higher in the newborn than in adults, while the expression of pro-inflammatory factors is generally lower than that of adults.
Conclusion (1) the change of TLR signaling pathway in umbilical cord blood induced by LPS is significantly different from that in adults. (2) the gene expression characteristics of TLR signaling pathway in newborn infants are negatively regulated, which may be one of the important mechanisms of its anti infection immune response.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R722

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