【作者】 张园园；

【导师】 陈志敏；

【作者基本信息】 浙江大学 ， 儿科学（专业学位）， 2014， 博士

【摘要】 第一部分难治性肺炎支原体肺炎的临床特征研究背景与目的：近年来临床观察与研究发现,肺炎支原体(Mycoplasma pneumoniae, MP)感染引起的难治病例明显增多,部分患者应用大环内酯类抗生素治疗后病情仍未见好转,不但引起胸腔积液、肺不张、肺实变、胸膜增厚等肺内并发症,而且还会引起一系列肺外并发症,甚至致死性肺炎。因此,如何早期识别难治性肺炎支原体肺炎(refractory Mycoplasma pneumoniae pneumonia, RMPP),并且把握治疗时机进行合理的治疗,成为支原体肺炎研究领域的一个热点课题。本研究旨在探讨RMPP的临床特征及其发病的独立相关因素,为临床医师早期识别RMPP,并且把握时机进行合理治疗提供依据。方法：通过对2011～2013年我院住院的708例肺炎支原体肺炎(Mycoplasma pneumoniae pneumonia, MPP)患儿临床资料的回顾性分析,比较普通MPP和RMPP患儿在临床症状、实验室结果和影像学特征等方面的差异,并将两组间差异有统计学意义的实验室指标作ROC曲线,寻找预示RMPP的危险因素。结果：1.708例MPP患儿中,普通MPP508例(508/708,71.8%),RMPP200例(200/708,28.2%),RMPP组患儿中位年龄明显大于普通MPP组患儿[5.6(3.5～7.8)岁vs3.3(1.8～6.0)岁,P<0.001]。2. RMPP组患儿发热、高热、胸闷、气促和肺部呼吸音减低阳性率分别为100%、98.5%、3.5%、14.0%和37.5%,高于普通MPP组(分别为84.3%、71.1%、0.4%、7.9%和37.5%)(P均<0.05)。3.普通MPP组入院前中位发热天数、总发热天数和住院天数分别为7(3-9)天、7(3-10)天和6(4-7)天,而RMPP组分别为10(8-12)天、14(12～17)天和9(7-12)天,差异具有明显的统计学意义(P<0.001)。RMPP组肺外并发症的发生率(40.0%)明显高于普通MPP组(20.5%),两组间差异具有明显统计学意义(P<0.001)。4.比较RMPP组和普通MPP患儿在外周血中性粒细胞百分比%[71.5(61.4～78.9) vs55.3(42.6～65.4)]、C反应蛋白(mg/L)[28(9-81) vs5(0-12)]、前降钙素(ng/ml)[0.15(0.07～0.34) vs0.06(0.03-0.12)]、乳酸脱氢酶(IU/L)[508(384-666) vs368(311～458)]、IgM (g/L)[1.91(1.30～2.58) vs1.58(1.18～2.19)]、IgA (g/L)[1.14(0.75-1.62) vs0.82(0.51～1.31)]、CD8+%[23.40(18.37～30.20) vs21.61(17.12～26.10)]、IL-6(pg/ml)[32.30(12.50～80.20) vs8.20(4.30～21.93)]、IL-10(pg/ml)[7.10(4.60～10.25) vs4.30(3.10～6.48)]、IFN-y (pg/ml)[11.75(6.80～36.73) vs7.90(4.13～12.10)]等指标,RMPP组均明显高于普通MPP组(P均<0.01)；而淋巴细胞百分比[18.9(12.9～26.8) vs35.1(24.5～47.4)]和CD4+/CD8+[1.60(1.23～2.13) vs1.43(1.17～1.90)]较普通MPP组明显下降(P<0.01)。5. RMPP组影像学表现以大片病灶为主(68%),普通MPP组则以斑片影为主(62.8%),两组有明显统计学差别P<0.001)。RMPP患儿肺内并发症如胸腔积液、肺不张、肺实变、坏死性肺炎的发生率分别为42.5%、23.5%、19%和2.5%,明显高于普通MPP患儿(分别为9.4%、9.4%、3.3%、0.2%)(P均<0.01)。6. RMPP组患儿需吸氧或需BAL的比例分别为20.0%和69.0%,明显高于普通MPP组患儿5.9%和25.2%,其差异具有明显统计学意义(P<0.001)。7.当MPP患儿出现血N%≥66.1%, L%≤23.4%, CRP≥12.5mg/L, PCI≥0.125ng/ml, LDH≥437IU/L, IL-6≥10.65pg/ml, IL-10≥5.65pg/ml时,应注意该MPP可能为难治性。结论：1.RMPP患儿平均年龄明显大于普通MPP组患儿,并具有更严重的临床表现、实验室异常指标和胸部影像学表现,并发症的发生率亦明显高于普通MPP组患儿,更需要吸氧及BAL等治疗。2.当MPP患儿出现血N%≥66.1%, L%≤23.4%, CRP≥12.5mg/L, PCT≥0.125ng/ml, LDH≥437IU/L, IL-6≥10.65pg/ml, IL-10≥5.65pg/ml时,应注意该MPP可能为难治性。第二部分支气管肺泡灌洗对儿童难治性肺炎支原体肺炎合并肺部大片病灶的疗效探讨研究背景与目的：纤维支气管镜和镜下支气管肺泡灌洗(Bronchoalveolar lavage, BAL)是近些年来儿童呼吸系统疾病诊治中安全、有效的手段,可以通过纤维支气管镜直视病变部位,观察支气管粘膜的损害情况,早期排除支气管内膜结核等其它疾病。同时,对于急性肺萎陷和肺不张等常规治疗无效的难治性病例,BAL能迅速清除气道内粘稠分泌物和粘液栓,使气道恢复通畅,肺组织复张,改善肺通气和换气功能。到目前为止,尚未有关于BAL在儿童难治性肺炎支原体肺炎(refractory Mycoplasma pneumoniae pneumonia, RMPP)中疗效的前瞻性研究。本研究旨在探讨BAL治疗合并肺部大片病灶的RMPP患儿的疗效,并探讨BAL应用时机对疗效的影响。方法：将2013年1月1日至2013年6月30日至本院呼吸科住院治疗的RMPP合并肺部大片病灶的患儿作为研究对象,共有35例患儿完成整个研究过程。根据BAL的时间,22例患儿进入早期干预组(入院后1-3天行BAL)；13例进入晚期干预组(入院后4～12天行BAL)。比较两组患儿的临床症状、主要实验室及影像学结果的恢复情况。结果：1.经BAL后,患儿的临床症状均得到改善,所有患儿在BAL后24小时内体温高峰下降,28例(80%)患儿在BAL后2天内体温恢复正常。BAL后中位发热时间为1(0-2)天。2.早期干预组入院后中位发热天数和中位住院天数分别为4(3～5)天和7(6～8)天,而晚期干预组入院后中位发热天数和中位住院天数分别为5(4-6)天和10(7-12)天,差异具有统计学意义(P<0.05)。3.早期干预组WBC和CRP恢复正常天数分别为6(3-7)天和6(3-7)天,较晚期干预组的8(7～11)天和8(6-11)天明显缩短,两者存在明显统计学差异(P<0.01)。4.入院7天胸部影像学检查发现,早期干预组胸片好转明显优于晚期干预期,前者有66.7%的肺不张复张,后者仅14.3%(P<0.05),具有统计学差异；前者胸腔吸收率为54.5%,后者为14.3%,但未达统计学差异(P>0.05)。5.在所有患儿BAL操作过程中,均无不良事件发生。结论：对于RMPP合并肺部大片病灶的患儿,在常规治疗的基础上,给予BAL能够缩短病程,促使炎症吸收,利于肺不张复张,其疗效确切,安全性高；早期应用疗效更好。第三部分TIPE2对MP感染的负向调控作用及其机制研究研究背景与目的：由于肺炎支原体(Mycoplasma pneumoniae, MP)感染临床表现多样,易被漏诊或误诊,且儿童处于生长发育阶段,限制了喹诺酮类等抗菌药物的使用,给治疗工作带来一定的困难,所以探索调控MP感染的机制,可能为寻求治疗MP感染的新方法提供理论基础和研究方向。肿瘤坏死因子α诱导蛋白8样分子2(tumor necrosis factor-a-induced protein8-like2, TIPE2)为近年来发现的在Toll样受体(toll-like receptor, TLR)信号通路中起负向调节作用的重要免疫分子,能抑制过强的免疫应答。而TLRl/2/6信号通路在MP感染的免疫反应中起重要作用,于是我们推测TIPE2可能会参与MP感染效应。然而TIPE2对MP感染的调控,尤其是TIPE2对MP感染机体的信号通路的调控,以及TIPE2如何调控信号通路尚未有研究报道。本研究旨在探讨了TIPE2对MP感染的调控作用其及相关分子机制。方法：临床研究对象为2012年6月至2013年6月因临床确诊MPP在我院治疗的患儿130例,并根据临床表现将MPP患儿分为普通MPP(72例)和RMPP(58例),抽取外周血检测其TIPE2表达的变化,并以60例健康体健儿童作为对照。同时在体外细胞水平检测了MP感染后巨噬细胞TIPE2的表达变化。并通过干扰实验研究TIPE2对MP感染的调控作用,通过Western-blot实验探索其如何调控信号通路。结果：1. TIPE2在人外周血单核细胞中有表达,MP感染患儿外周血单核细胞中TIPE2mRNA表达较健康儿童下调,且难治型患儿下调更明显。2.MP刺激巨噬细胞可以降低TIPE2mRNA及其蛋白的表达。MP感染同样能够引起小鼠巨噬细胞系Raw264.7和小鼠腹腔来源的巨噬细胞TIPE2明显的下调。3.MP刺激巨噬细胞可引起炎性细胞因子IL-6、TNF-α、IL-1β的分泌。4.采用siRNA497干扰序列进行实验,结果发现,干扰内源性TIPE2的表达显著增强了MP刺激产生的IL-6、TNF-α、IL-1β的表达和蛋白分泌。5.干扰内源性TIPE2能明显增加MP诱导的ERK、JNK和P38的活化。结论：1.MP感染可引起巨噬细胞中TIPE2表达的下调,且与疾病严重程度呈负相关。2. TIPE2可负向调控MP感染引起的炎性细胞因子的分泌。3. TIPE2可能通过抑制MAPK信号的活化,负向调控MP感染引起的炎性细胞因子的分泌。更多还原

【Abstract】 Part I Clinical Characteristics of Refractory Mycoplasma Pneumoniae PneumoniaObjective:Although MP infection was traditionally thought to be a self-limited process, more and more severe cases even fatal cases of MP infections were reported in recent years, it may sometimes cause various pulmonary and extra-pulmonary complications, such as pleural effusion, pulmonary atelectasis, consolidation, encephalitis, pericarditis and anemia, and progress to a severe life-threatening pneumonia. So it is important for physicians to recognize refractory Mycoplasma pneumoniae pneumonia (RMPP) earlier and give proper therapy. The aim of this study is to analyze the clinical characteristics of RMPP, and study the critical values of the independent correlation factors of RMPP.Methods:Retrospective analysis was performed on the clinical data of708children with Mycoplasma pneumoniae pneumonia (MPP). The patients were divided into "general MPP group" and "RMPP group" according to the signs and symptoms. The clinical manifestations, laboratory examination, radiological features of the children were compared. In order to seek the reference indexes which indicate refractoriness, an ROC curve was made, of which the diagnostic cut-off was seven independent correlation factors while grouping was made.Results:The general MPP group had508children (508/708,71.8%) while MPP group had200(200/708,28.2%). The RMPP group children had the following performances:The age of children in RMPP group were older than that in general MPP group (P<0.001). The incidence of fever, high-grade fever, chest tightness, tachypnea, decrease in breath sound and extra-pulmonary complications were significantly higher compared to those in general MPP group, which needs more oxygen and BAL therapy. The median fever duration prior to admission was7(3-9) days and10(8-12) days, the median total fever duration was7(3～10) days and and14(12～17) days, and the median hospitalization duration was6(4-7) days and9(7-12) days, respectively in the general MPP group and the RMPP group (P<0.001). The levels of C-reactive protein (CRP), procalcitonin (PCT), actic dehydrogenase (LDH), immunoglobulin M (IgM), IgA, interleukin (IL)-6, IL-10, IFN-y and the percentage of neutrophils, CDs+in RMPP group were significantly higher than those in general MPP group (P<0.001); while the percentage of lymphocytes and the ratio of CD4+to CD8+were lower than those in general MPP group (P<0.001). Radiological manifestations:large lesions and pulmonary complications, such as pleural effusion, atelectasis, consolidation and necrotizing pneumonia were more frequently seen in RMPP group than those in general MPP group (P<0.001). The critical values of the seven independent correlation factors were N%66.1%, L%3.4%, CRP12.5mg/L, PCT0.125ng/ml, LDH437IU/L, IL-610.65pg/ml and IL-105.65pg/ml.Conclusions:Children with RMPP are older than children with general MPP, and RMPP may lead to more complications, severe clinical features, radiological manifestations, and needs more oxygen and BAL therapy. MPP maybe progress into RMPP in some situations such as N%≥66.1%, L%≤23.4%, CRP>12.5mg/L, PCT≥0.125ng/ml, LDH>437IU/L, IL-6≥10.65pg/ml and IL-10>5.65pg/ml. Part II Effects of Early Bronchoalveolar Lavage on Refractory Mycoplasma Pneumoniae Pneumonia with Large Pulmonary Lesions in ChildrenObjective:Nowadays, flexible bronchoscopy (FOB) with bronchoalveolar lavage (BAL) is an important and indispensable tool to assess the structure and function of lower airways, diagnose pulmonary infiltrative lesion in pediatric patients. Furthermore, endobronchial tuberculosis could be excluded earlier. Additionally, FOB could also perform therapeutic interventions, such as treating acute lung collapse and atelectasis which was refractory to conventional therapy. To our knowledge, this is the first study to focus on the effect of BAL in RMPP patients in a prospective fashion. The aim of this study is to prospectively evaluate the effect of early BAL on refractory Mycoplasma pneumoniae pneumonia (RMPP) with radiologically proved large pulmonary lesions in children.Methods:A total of35children diagnosed as RMPP with radiologically proved large pulmonary lesions completed the study. According to the time point of BAL, they were divided into two groups. The early BAL group:BAL was performed within three days after admission (n=22), and the late BAL group:BAL was performed three days later after admission (n=13). Clinical effect was compared between these two groups.Results:After BAL therapy, improvement in clinical symptoms, laboratory findings as well as resolution of pulmonary lesion on radiography were obtained in all subjects. The median fever duration after admission was4(3-5) days and7(6-8) days (P<0.05), and the median hospitalization duration was5(4-6) days and10(7-12) days (P><0.05), respectively in the early BAL group and the late BAL group. About7days after admission,66.7%of subjects in the early BAL group showed resolution of atelectasis on X-ray image versus a corresponding rate of14.3%in the late BAL group (P<0.05). Furthermore, laboratory indices recovered quicker in the early BAL group than those in the late BAL group (P<0.01).Conclusions:BAL appeared to be efficacious and well-tolerated treatment for RMPP with radiologically proved large pulmonary lesion, and early application of BAL may be more beneficial. Part Ⅲ The negative role and its underlying mechanism of TIPE2on Mycoplasma Pneumoniae infectionObjective:The morbidity and mortality of Mycoplasma Pneumoniae (MP) infection increased annually, comprising a serious threat to children’s health. Recently, studies showed that TLR signaling pathways played an important role in inflammation induced by mycoplasma pnuemonia. Tumor necrosis factor-a-induced protein8-like2(TIPE2), a member of the tumor necrosis factor-a-induced protein-8(TNFAIP8) family is a negative regulator of TLR, and could prevent hyperresponsiveness and maintain immune homeostasis. However, the effect and underlying mehcanisms of TIPE2on MP infection remain unclear. This study will investigate the influence of MP infection on the protein levels of TIPE2expression, and explore the effect and its underlying mechanisms regulating MP infection response by TIPE2.Methods:We collected the peripheral blood monouclear cells from130children with Mycoplasma Pneumoniae pneumonia (MPP), which were divided into general MPP group and Refractory Mycoplasma Pneumoniae pneumonia (RMPP) group according to the clinical manifestation, and detected the levels of TIPE2mRNA. We also detected the levels of TIPE2in macrophages in vitro after MP infection. Meanwhile, we interfered the TIPE2expression in macrophage, study the production of cytokines, such as TNF-a, IL-6and IL-1β, and detected the changes of signal pathway after MP infection.Results:In our study, we found that TIPE2were downregulated in peripheral blood monouclear cells of children with MP infection, which were correlated with the degree of severity of MPP. In vitro, we also found the expression of TIPE2was downregulated and cytokines, such as TNF-a, IL-6and IL-1β were produced after MP infection. Through loss of function, we found the production of TNF-a, IL-6and IL-1β were upregulated, and the signal pathway of MAPK was activated.Conclusions:Our data showed that TIPE2were downregulated in peripheral blood monouclear cells of children with MP infection and in macrophages in vitro, and knockdown of TIPE2in macrophage significantly promoted the production of TNF-a, IL-6and IL-1β through inhibiting the MAPK signal pathway. The results will provide insights into the molecular events controlling the MP infection.更多还原