半胱氨酰白三烯影响OSAHS患儿腺样体T细胞增殖及凋亡的作用与机制

发布时间：2018-06-23 10:40

本文选题：孟鲁司特钠 + 鼻喷糖皮质激素　；参考：《重庆医科大学》2014年博士论文

【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。
[Abstract]:Part one: clinical observation of montelukast sodium and nasal corticosteroids in the treatment of OSAHS in children
Objective: to analyze the clinical characteristics of children with obstructive sleep apnea hypopnea syndrome (obstructive sleep apnea hypopnea syndrom, OSAHS), and to compare the clinical efficacy of cysteinyl leukotriene receptor 1 (CysLTR1) blocker montelukast sodium and nasal glucocorticoid in the treatment of children OSAHS. Objective to investigate the feasibility of non operative treatment of mild OSAHS in children.
Methods: (1) retrospective analysis of 2257 cases of OSAHS patients in our hospital from January 2011 to September 2013, and analyzed the patients' sex, age, history of past diseases, tonsillar adenoids, PSG monitoring data AHI and the lowest SpO2, and (2) selected from October 2012 to 2013 according to the inclusion and exclusion criteria. In October, 189 children with mild OSAHS were diagnosed and randomly assigned to three treatment groups: group A (oral montelukast) 63 cases, group B (nasal spray furfuric acid Momison) 63 cases, group C (oral montelukast sodium plus nasal spray furfuric acid) 63 cases; (3) the treatment regimen was as follows: A group: mouth: mouth Take montelukast sodium (6 years old 4mg, 6 years old 5mg) 1 / a night for 12 weeks; group B: nasal spray Mometasone Furoate Nasal Spray 1 times / morning, left and right nasal spray (50 g) for 12 weeks; group C: oral montelukast sodium (use with A), nasal spray (used with A group) for 12 weeks; (4) using a telephone questionnaire The clinical symptoms were followed up and the PSG monitoring was performed before and after 12 weeks of treatment. (5) the improvement and efficiency of clinical symptoms before and after treatment in the three groups were compared and analyzed. (6) the treatment group was divided into group A1 (effective group) and group A2 (treatment group), and the difference of the clinical features of the two groups was compared and analyzed.
Results: (1) in hospitalized children with OSAHS, 3-6 years of age before school age were the peak of onset (56%), more males than women (1.6:1), moderate (52.5%), mild children in a certain proportion (32.3%), some patients with allergic rhinitis (11.7%); (2) after treatment, three groups of children snoring, sleep unease, and apnea, and other clinical symptoms and PSG The monitoring results were obviously improved compared with those before the treatment. (3) compared with group A and B group, the time of disappearance of snoring, apnea and sleep uneasiness in group C was shorter. The total effective rate of group C was higher than that of group A and B group, and there was no significant difference between group A and B group, and (4) the size of tonsil in group A2 was significantly higher than that of A1 group.
Conclusions: (1) the proportion of mild OSAHS in children with OSAHS is about 1/3; (2) for children with mild OSAHS, oral montelukast or nasal spray glucocorticoids or two combinations can be used in the perioperative period, which can effectively reduce the volume of adenoids and relieve some of the clinical symptoms. The total effective rate of the combined use is higher than that of the single drug, and the symptoms can be relieved faster. The above results suggest that the inflammatory reaction may be involved in the pathological mechanism of hypertrophy of upper airway lymphatic tissue; (3) montelukast sodium may be not effective in the treatment of OSAHS with more than 3 degrees of tonsillar hypertrophy.
The second part is the effect of LTD4 on proliferation and apoptosis of adenoid T cells.
Objective: To observe the expression and localization of CysLTR1 in adenoid tissue of children with OSAHS, and to study the effect of LTD4 on the proliferation and apoptosis of CD4+T and CD8+T cells in adenoid mononuclear cells (AdMC).
Methods: (1) collect adenoidoid tissue in children with adenoidectomy OSAHS or secretory otitis media (control group) in the Affiliated Children's Hospital of Medical University Of Chongqing; (2) the collected specimens were divided into three groups: AHI < 5, 5 < AHI < 10, AHI > 10, AdMC, ConA stimulation for 48 hours, the collection of supernatant and ELISA examination. The expression level of LTC4 was measured; (3) the specimens were collected to prepare paraffin sections, and the expression of CysLTR1 in the adenoid tissue was detected by immunohistochemistry. The double immunofluorescent laser confocal laser confocal microscopy was used to observe whether CysLTR1 could be expressed in CD3+T cells; (4) the separation of AdMC was divided into 4 groups: against group, PHA stimulation group, PHA+LTD4 group and PHA+LTD4+ montrenster. (montelukast) group, CFSE labeled flow cytometry (FCM) was used to detect the regulation of LTD4 on CD4+ and CD8+T cell proliferation in AdMC stimulated by PHA; (5) isolated AdMC, divided into 4 groups: control group, DEX stimulation group, DEX+LTD4 group, DEX+LTD4 group Regulating effect.
Results: (1) the expression level of adenoid CysLTR1 in the OSAHS group was significantly higher than that in the control group. CysLTR1 was partially expressed in CD3+T cells in the adenoid tissues of children with OSAHS, but not the dominant expression; (2) the level of LTC4 secreted in the medium and severe groups was significantly higher than that in the normal control group and the mild group; (3) LTD4+PHA after AdMC, CD4+T and CD8+T cells were found. The proliferation rate was significantly increased than that of the PHA group. Montelukast could inhibit the proliferation promoting effect of LTD4. (4) after LTD4+DEX co stimulated AdMC, the early apoptosis rate of CD4+T and CD8+T cells was significantly lower than that of DEX group, and montelukast inhibited the inhibitory apoptosis effect of LTD4.
Conclusions: (1) the expression of LTC4 in AdMC supernatant is related to the degree of OSAHS in children. (2) the expression of CysLTR1 in adenoid tissue is higher and partial CysLTR1 is located in CD3+T cells, suggesting that CysLTs may be associated with hyperplasia and hypertrophy of adenoid tissue, and may be involved in the pathogenesis of OSAHS in children; (3) LTD4 to CD4+T and CD8+T cells from AdMC origin. Proliferation and apoptosis have regulatory function.
The third part is the mechanism of LTD4 regulating the proliferation and apoptosis of T cells.
Objective: To investigate whether LTD4 regulates proliferation and apoptosis of CD4+T cells and CD8+T cells in AdMC through MAPKs pathway.
Methods: (1) separation of AdMC, divided into control group and treatment group, the treatment group added PHA (concentration 10ug/ml) and LTD4 (concentration 1 x 10-4mmol/l), 24 orifice plates, 37 centigrade, 5%CO2 incubator culture. According to the time point 5min, 15min, 30min, 1H, 2h, 12h cells, extract the total egg white; (2) detect and detect the phosphorylated eggs The effect of Rhizoma Bletillae non phosphorylated total protein; (3) separation of AdMC, divided into four groups: control group, PHA group, PHA+LTD4 group and PHA+LTD4+p38 inhibitor SB203580 group or PHA+LTD4+ERK1/2 inhibition PD98059 group, CFSE labeling FCM detection, observe the value of CD4+T cell and CD8+T cell change; (4) separate AdMC, divided into four groups: control group D4 group and DEX+LTD4+p38 inhibitor SB203580 group or DEX+LTD4+ERK1/2 inhibiting PD98059 group and Annexin V FITC/PI double stained FCM were detected to observe the changes of early apoptosis rate of CD4+T cells and CD8+T.
Results: (1) Western Blot assay showed that in group PHA+LTD4, after LTD4 stimulated AdMC after 5min, the phosphorylation level of p38 increased significantly (P < 0.05). After 1 hours of stimulation, the expression amount reached the peak (P < 0.001). After stimulation for 2 hours, the expression decreased gradually. After stimulation for 12 hours, the expression was reduced to the base level, and the expression of non phosphorylated total protein of p38 was not expressed. Significant changes (P > 0.05).CysLTR1 inhibitor montelukast or p38 inhibitor SB203580 could inhibit the phosphorylation reaction. (2) the level of ERK1/2 phosphorylation level was significantly increased after LTD4 stimulation of 30min (P < 0.01). After 1 hours of stimulation, the expression amount reached the peak (P < 0.001). After 1.5 hours of stimulation, the expression decreased gradually and stimulated 2 hours. After that, the expression level was basically reduced to the basic level, and the expression of ERK1/2 non phosphorylated total protein was not significantly changed at all time points (P > 0.05).CysLTR1 inhibitor montelukast or ERK1/2 inhibitor PD98059 could inhibit the phosphorylation reaction. (3) LTD4 has no significant effect on the phosphorylation of JNK pathway. (4) CFSE labeling method FCM detection shows that PD98059 can be effective Inhibition of LTD4 regulated PHA induced proliferation of CD4+T cells and CD8+T cells in ADMC.
Conclusions: (1) LTD4 can activate the ERK1/2 and p38MAPK pathways in AdMC, increase the phosphorylation level, but have no significant effect on the JNK pathway. (2) the ERK1/2 signaling pathway may be one of the downstream signaling pathways of LTD4 regulating CD4+T and CD8+T cells in AdMC of children with OSAHS. (3) the pathway is activated in children with OSAHS. It did not significantly affect the effect of LTD4 on proliferation and apoptosis, and what role p38MAPK played in it needs further study.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R766

【参考文献】