【作者】 舒艳；

【导师】 刘恩梅；

【作者基本信息】 重庆医科大学 ， 儿科学， 2014， 博士

【摘要】 第一部分孟鲁司特钠与鼻用糖皮质激素治疗儿童OSAHS的临床疗效观察目的：分析诊断为儿童阻塞性睡眠呼吸暂停低通气综合征(obstructive sleep apnea hypopnea syndrom，OSAHS)并住院行手术治疗的患儿临床特征，对比观察半胱氨酰白三烯受体1（CysLTR1）阻断剂孟鲁司特钠和鼻用糖皮质激素治疗儿童OSAHS的临床疗效，探讨临床非手术治疗儿童轻度OSAHS的可行性。方法：（1）回顾性分析2011年1月至2013年9月在我院耳鼻咽喉科就诊并住院手术的2257例OSAHS病人资料，分析病人的性别、年龄、既往疾病史、扁桃体腺样体特征、PSG监测数据AHI和最低SpO2等临床特征；（2）按照纳入与排除标准选取2012年10月至2013年10月在我院耳鼻咽喉科专科门诊就诊并诊断为OSAHS轻度的患儿189例，随机分配入三个治疗组：A组（口服孟鲁司特钠治疗）63例，B组（鼻喷糠酸莫米松治疗）63例，C组（口服孟鲁司特钠+鼻喷糠酸莫米松治疗）63例；（3）治疗方案如下：A组：口服孟鲁司特钠（＜6岁者4mg，≥6岁者5mg）1次/晚，连续12周；B组：鼻喷糠酸莫米松鼻喷雾剂1次/晨起，左右鼻腔各一喷（50μg），连续12周；C组：口服孟鲁司特钠(用法同A组)，鼻喷糠酸莫米松鼻喷雾剂(用法同A组)，连续12周；（4）采用电话问卷式调查随访临床症状，并评分；治疗前和治疗12周后行PSG监测；（5）对比分析三组病人治疗前后临床症状改善情况及有效率；（6）将治疗A组分为A1组（治疗有效组）和A2组（治疗无效组），对比分析两组临床特征的差异性。结果：（1）住院OSAHS患儿中，学龄前3-6岁是发病高峰(56%)，男多于女(1.6:1)，病情以中度为主((52.5%)，轻度的患儿占有一定比例(32.3%)，部分病人伴有变应性鼻炎(11.7%)；（2）治疗后，三组患儿打鼾、张口呼吸、睡眠不安、及呼吸暂停等临床症状及PSG监测结果均较治疗前明显改善；（3）C组患儿与A组和B组相比，打鼾、呼吸暂停及睡眠不安等临床症状消失时间均缩短，C组患儿治疗后总有效率较A组和B组均高，A组和B组两组间各项值无显著差异；（4）A2组的扁桃体大小分级明显高于A1组。结论：（1）轻度OSAHS在住院OSAHS患儿中占有约1/3比例；（2）对轻度OSAHS患儿，围手术期可采用口服孟鲁司特钠或鼻喷糖皮质激素或二者联用治疗，能有效缩小腺样体体积，缓解部分临床症状，联合用药总有效率较单用药更高，缓解症状更快。以上临床观察结果均提示炎症反应可能参与了上气道淋巴组织增生肥大的病理机制；（3）孟鲁司特钠可能对伴有3度以上扁桃体肥大的OSAHS治疗效果不佳。第二部分LTD4对腺样体T细胞增殖及凋亡的作用研究目的：观察CysLTR1在OSAHS患儿腺样体组织中的表达与细胞定位，研究LTD4对腺样体单个核细胞(AdMC)中CD4+T和CD8+T细胞增殖和凋亡的影响。方法：（1）收集重庆医科大学附属儿童医院耳鼻咽喉科因OSAHS或分泌性中耳炎（对照组）住院行腺样体切除术患儿的腺样体组织；（2）收集的标本分为三个组：AHI＜5组、5＜AHI＜10组、AHI＞10组，分离AdMC，用ConA刺激培养48小时，收集上清，ELISA检测LTC4的表达水平；（3）收集的标本制备石蜡切片，免疫组化法检测CysLTR1在腺样体组织中的表达情况，免疫荧光双标法激光共聚焦观察CysLTR1是否可表达于CD3+T细胞；（4）分离AdMC，分为4个组：对照组，PHA刺激组，PHA+LTD4组和PHA+LTD4+孟鲁斯特纳（montelukast）组，CFSE标记法流式细胞术（FCM）检测LTD4对PHA刺激的AdMC中CD4+、CD8+T细胞增殖的调节作用；（5）分离AdMC，分为4个组：对照组，DEX刺激组，DEX+LTD4组，DEX+LTD4组+montelukast组，Annexin Ⅴ FITC/PI双染FCM检测LTD4对DEX诱导的AdMC中CD4+、CD8+T细胞凋亡的调节作用。结果：（1）OSAHS患儿组腺样体CysLTR1的表达水平显著高于对照组，CysLTR1在OSAHS患儿腺样体组织中部分表达于CD3+T细胞，但并非优势表达；（2）中重度组分泌的LTC4水平显著高于正常对照组与轻度组；（3）LTD4+PHA共同刺激AdMC后，CD4+T和CD8+T细胞的增殖率比PHA组明显增加，montelukast可抑制LTD4的促增殖效应；（4）LTD4+DEX共同刺激AdMC后，CD4+T和CD8+T细胞的早期凋亡率比DEX组明显降低，montelukast可抑制LTD4的抑制凋亡效应。结论：（1）AdMC上清液中LTC4的表达与OSAHS患儿病情程度相关；（2）CysLTR1在腺样体组织中表达增高，部分CysLTR1定位于CD3+T细胞，提示CysLTs可能与腺样体组织增生肥大相关，并可能参与儿童OSAHS的发病机理；（3）LTD4对AdMC来源的CD4+T和CD8+T细胞的增殖和凋亡具有调节功能。第三部分LTD4调节腺样体T细胞增殖及凋亡的机制研究目的：探讨LTD4是否通过MAPKs通路调节AdMC中CD4+T细胞和CD8+T细胞细胞增殖和凋亡。方法：（1）分离AdMC，分为对照组和处理组，处理组中加入PHA（浓度10ug/ml）和LTD4(浓度1×10-4mmol/l)，24孔板中，37℃，5%CO2孵箱培养。按培养时间点5min、15min、30min、1h、2h、12h收集细胞，提取总蛋白；（2）Western blot法检测LTD4对MAPK通路P38、ERK1/2、JNK的磷酸化蛋白及非磷酸化总蛋白的影响；（3）分离AdMC，分为四个组：对照组、PHA组、PHA+LTD4组和PHA+LTD4+p38抑制剂SB203580组或PHA+LTD4+ERK1/2抑制PD98059组，CFSE标记法FCM检测，观察CD4+T细胞和CD8+T细胞增值率的变化；（4）分离AdMC，分为四个组：对照组、DEX组、DEX+LTD4组和DEX+LTD4+p38抑制剂SB203580组或DEX+LTD4+ERK1/2抑制PD98059组，Annexin Ⅴ FITC/PI双染FCM检测，观察CD4+T细胞和CD8+T早期凋亡率的变化。结果：（1）Western Blot法检测显示，PHA+LTD4组中，LTD4刺激AdMC后5min后，p38磷酸化水平明显开始增加（P＜0.05)，刺激1小时后，表达量达高峰（P＜0.001)，刺激2小时后，表达量逐渐下降，刺激12小时后，表达量降至基础水平，而p38非磷酸化总蛋白的表达无明显变化（P＞0.05)。CysLTR1抑制剂montelukast或p38的抑制剂SB203580可抑制该磷酸化反应。（2）PHA+LTD4组中，LTD4刺激30min后，ERK1/2磷酸化水平表达水平显著增加（P＜0.01)，刺激1小时后，表达量达高峰（P＜0.001)，刺激1.5小时后，表达量逐渐下降，刺激2小时后，表达量基本降至基础水平，而ERK1/2非磷酸化总蛋白的表达在各时间点均无明显变化（P＞0.05)。CysLTR1抑制剂montelukast或ERK1/2的抑制剂PD98059可抑制该磷酸化反应。（3）LTD4对JNK通路的磷酸化无显著影响。（4）CFSE标记法FCM检测显示，PD98059能有效抑制LTD4调节的PHA诱导的ADMC中CD4+T细胞和CD8+T细胞增殖。结论：（1）LTD4可激活AdMC中ERK1/2和p38MAPK通路，使其磷酸化水平增加，但对JNK通路无显著影响。（2）ERK1/2信号通路可能是LTD4调节OSAHS患儿AdMC中CD4+T细胞和CD8+T细胞增殖的下游信号通路之一。（3）在OSAHS患儿AdMC中，LTD4激活了p38MAPK通路，但p38MAPK通路并未显著影响LTD4调节增殖和凋亡的效应，p38MAPK究竟发挥了怎样的作用还需进一步研究。更多还原

【Abstract】 PART ⅠTHE CLINICAL EFFECTS OF MONTELUKAST ANDINTRANASAL CORTICOSTEROIDS IN CHILDRENOSAHSObjective: Analysis clinical features in children with OSAHS inhospital. Comparative analysis the clinical effects of a cysteinyl LT (cysLT)receptor1antagonist (montelukast) and intranasal corticosteroid (ICS).Discuss the feasibility of non-surgical treatment in children with mildOSAHS.Methods:(1) Retrospective analysis the data of children withOSAHS at department of otorhinolaryngology in CHCMU from January2011to September2013. The gender, age, diseases history, characteristic oftonsil and adenoid, AHI and nadir SpO2were analyzed;(2) One hundredand eight-nine patients with mild OSAHS were divided into3groups (thegroup A, the group B and the group C) randomly from October2011toOctober2012.(3) Therapeutic schedule:63were treated with oralmontelukast (OM) in group A for12weeks.63were treated with ICS ingroup B for12weeks.63were treated with a combination of OM and ICSin group C for12weeks.(4) Clinic symptoms and polysomnographicchanges in polysomnographic findings were followed with telephoneinquiring.(5) Contrastive analysis was applied to the analysis of improvement of symptoms and effective rate.(6) The group A was devidedinto the group A1(effective therapy group)and the group A2(invalidtherapy group). The differences of clinical features of the two groups wereanalyzed.Results:(1) There was a peak prevalence at3-6years of age inChildren with OSAHS in hospital. The boys were more than girls inpatients. Though the children with moderate OSAHS were the most in allchildren, Children with mild occupiesed thirty-two point three percent.Some patients had the history of allergic disease such as allergichinitis(11.7%);(2) The clinical symptoms and PSG monitoring resultswere obviously improved posttreatment. The clinical symptoms includedsnoring, mouth breathing, restlessness, and sleep apnea;(3) The symptomsdisappeared more quickly in group C than group A or B. The total effectiverate in group C was higher than that of group A or B. significant differencewas not found between Group A and group B.(4) Tonsil size grading ingroup A2was obviously higher than that of group A1.Conclusion:(1) The children with mild OSAHS occupied theproportion about one-third in all hospitalized children with OSAHS.(2)The children with mild OSAHS may treated with OM and/or ICS inperioperative period. Non-surgical treatment can reduce the size of adenoidand Relieve part of clinical symptoms effectively. The total effective rate ofdrug combination is higher than that of single medication.The result showthat the inflammatory response may be involved in the pathologicalmechanism of airway lymphoid tissue hypertrophy.(3) The therapeuticeffect of the children with the larger tonsils is not good. PART ⅡTHE EFFECT OF LTD4ON THE PROLIFERATIONAND APOPTOSIS OF T CELL IN ADENOIDObjective: To observe CysLTR1expression in the adenoid inchildren with OSAHS, and investigate the effect of CysLTs on theproliferation and apoptosis of CD4+T and CD8+T cell in adenoid.Methods:(1) Collect the adenoid of the children undergoingAdenoidectomy.(2) The specimen were devided into3groups according tothe AHI (the control group: AHI＜5, the mild group:5＜AHI＜10and themoderate-to severe group:AHI＞10）. AdMC were separated and culturedusing96-well plates to exposed to ConA for48hours. The cell culturesupernatants were collected. The levels of LTC4in the supernatants of allgroups were determined using a human LTC4ELISA kit following themanufacturer’s instructions.(3) Adenoid were prepared for paraffinsections.Immunohistochemistry detected the expression of CysLTR1andImmunofluorescence assay located CysLTR.(4) AdMC were separated andrandomized to4groups: group A, control group; group B, PHA; group C,PHA+LTD4; and group D, PHA+LTD4+montelukast. FCM assay was usedto detect the CD4+T and CD8+T cell proliferation.(5) AdMC wererandomized to4groups: group A, control group; group B, DEX; group C,DEX+LTD4; and group D, DEX+LTD4+montelukast. FCM assay wasused to detect the CD4+T and CD8+T cell apopsis.Results:(1) Adenoid of children with OSAHS but not of controlsubjects have enhanced expression of CysLTR1. Part of CysLTR1immunoreactivity was detected in adenoid CD3+T cells.(2) moderate tosevere OSAHS show higher LTC4compared to mild OSAHS and controlsubjects.(3) The rate of increase of CD4+T and CD8+T cell in PHA+LTD4 group is higher than PHA group. Montelukast can inhibit the proliferationeffect.(4) The early apoptosis rate of CD4+T and CD8+T cell of DEX+LTD4group is lower than DEX group.Montelukast can inhibit the apopsiseffect.Conclusions:(1) Expression of LTC4is related to OSAHS severityin children.(2) The expression of CysLTR1enhanced markedly in adenoidchildren with OSAHS. Only part of CysLTR1immunoreactivity wasdetected in adenoid CD3+T cells. The result shows that CysLTR1may maybe related with adenoid hypertrophy and participate in the pathogenesis ofOSAHS in children.(3) LTD4can regulate the proliferation and apoptosis ofCD4+T and CD8+T cell in AdMC stimulated by PHA. PART ⅢMECHANISM OF EFFECT LTD4ON ROLIFERATION ANDAPOPTOSIS OF T CELL IN ADENOIDObjective: To explore whether LTD4by MAPKs pathway regulatedthe proliferation and apoptosis of CD4+T and CD8+T cells in AdMC.Methods:(1) AdMC were randomly assigned to2groups: group A,control group; group B: PHA10ug/ml+LTD41×10-4mmol/l. The cellswere grown in an incubator (37℃，5%CO2) and collected according to thetraining time for5min and15min,30min,1h,2h,12h and extracted theprotein.(2) Western Blot detected the expressions of p-p38、p-ERK1/2andp-JNK protein in ADMC treated with LTD4for each point in time.(3)AdMC were randomized to4groups: group A, control group; group B, PHA;group C, PHA+LTD4; and group D, PHA+LTD4+SB203580or PHA+LTD4+PD98059. FCM assay was used to detect change theproliferation of the CD4+T Cell and CD8+T cell.(4) AdMC wererandomized to4groups: group A, control group; group B, DEX; group C,DEX+LTD4; and group D, DEX+LTD4+SB203580orPHA+LTD4+PD98059. FCM assay was used to detect change the apoptosisof the CD4+T Cell and CD8+T cell.Results:(1) p-p38protein increased obviously after LTD4treated for5min （P＜0.05), and continued to the highest level for1hour （P＜0.001). The p38total protein expression had no obvious change. Thephosphorylation reaction was inhibited by the CysLTR1inhibitormontelukast or a selective p38MAP kinase inhibitor SB203580.(2)p-ERK1/2protein increased obviously after LTD4treated for15min（P＜0.01), and continued to the highest level for1hour（P＜0.001). Theexpression was down to the basic level for2hours. The ERK1/2totalprotein expression had no obvious change. The aforementioned three kindsof inhibitor can restrain the reaction also.(3) Obvious effect on p-JNK byLTD4were not observed.(4) The proliferation and apoptosis of CD4+T andCD8+T cell in adenoid mononuclear cells stimulated by PHA and LTD4were restrained by PD98059.Conclusion:(1) ERK1/2and p38MAPK pathway but no JNK wereactivated by LTD4.(2) LTD4promoted the proliferative effect of CD4+Tand CD8+T cell stimulated with PHA through ERK1/2pathway.(3)Effect of activated p38MAPK pathway by LTD4was not watched.更多还原