本文選題：孟魯司特鈉 + 鼻噴糖皮質(zhì)激素　； 參考：《重慶醫(yī)科大學(xué)》2014年博士論文

【摘要】：第一部分孟魯司特鈉與鼻用糖皮質(zhì)激素治療兒童OSAHS的臨床療效觀察 目的：分析診斷為兒童阻塞性睡眠呼吸暫停低通氣綜合征(obstructive sleep apnea hypopnea syndrom，，OSAHS)并住院行手術(shù)治療的患兒臨床特征，對比觀察半胱氨酰白三烯受體1（CysLTR1）阻斷劑孟魯司特鈉和鼻用糖皮質(zhì)激素治療兒童OSAHS的臨床療效，探討臨床非手術(shù)治療兒童輕度OSAHS的可行性。 方法：（1）回顧性分析2011年1月至2013年9月在我院耳鼻咽喉科就診并住院手術(shù)的2257例OSAHS病人資料，分析病人的性別、年齡、既往疾病史、扁桃體腺樣體特征、PSG監(jiān)測數(shù)據(jù)AHI和最低SpO2等臨床特征；（2）按照納入與排除標(biāo)準(zhǔn)選取2012年10月至2013年10月在我院耳鼻咽喉科�？崎T診就診并診斷為OSAHS輕度的患兒189例，隨機(jī)分配入三個治療組：A組（口服孟魯司特鈉治療）63例，B組（鼻噴糠酸莫米松治療）63例，C組（口服孟魯司特鈉+鼻噴糠酸莫米松治療）63例；（3）治療方案如下：A組：口服孟魯司特鈉（＜6歲者4mg，≥6歲者5mg）1次/晚，連續(xù)12周；B組：鼻噴糠酸莫米松鼻噴霧劑1次/晨起，左右鼻腔各一噴（50μg），連續(xù)12周；C組：口服孟魯司特鈉(用法同A組)，鼻噴糠酸莫米松鼻噴霧劑(用法同A組)，連續(xù)12周；（4）采用電話問卷式調(diào)查隨訪臨床癥狀，并評分；治療前和治療12周后行PSG監(jiān)測；（5）對比分析三組病人治療前后臨床癥狀改善情況及有效率；（6）將治療A組分為A1組（治療有效組）和A2組（治療無效組），對比分析兩組臨床特征的差異性。 結(jié)果：（1）住院OSAHS患兒中，學(xué)齡前3-6歲是發(fā)病高峰(56%)，男多于女(1.6:1)，病情以中度為主((52.5%)，輕度的患兒占有一定比例(32.3%)，部分病人伴有變應(yīng)性鼻炎(11.7%)；（2）治療后，三組患兒打鼾、張口呼吸、睡眠不安、及呼吸暫停等臨床癥狀及PSG監(jiān)測結(jié)果均較治療前明顯改善；（3）C組患兒與A組和B組相比，打鼾、呼吸暫停及睡眠不安等臨床癥狀消失時間均縮短，C組患兒治療后總有效率較A組和B組均高，A組和B組兩組間各項值無顯著差異；（4）A2組的扁桃體大小分級明顯高于A1組。 結(jié)論：（1）輕度OSAHS在住院OSAHS患兒中占有約1/3比例；（2）對輕度OSAHS患兒，圍手術(shù)期可采用口服孟魯司特鈉或鼻噴糖皮質(zhì)激素或二者聯(lián)用治療，能有效縮小腺樣體體積，緩解部分臨床癥狀，聯(lián)合用藥總有效率較單用藥更高，緩解癥狀更快。以上臨床觀察結(jié)果均提示炎癥反應(yīng)可能參與了上氣道淋巴組織增生肥大的病理機(jī)制；（3）孟魯司特鈉可能對伴有3度以上扁桃體肥大的OSAHS治療效果不佳。 第二部分LTD4對腺樣體T細(xì)胞增殖及凋亡的作用研究 目的：觀察CysLTR1在OSAHS患兒腺樣體組織中的表達(dá)與細(xì)胞定位，研究LTD4對腺樣體單個核細(xì)胞(AdMC)中CD4+T和CD8+T細(xì)胞增殖和凋亡的影響。 方法：（1）收集重慶醫(yī)科大學(xué)附屬兒童醫(yī)院耳鼻咽喉科因OSAHS或分泌性中耳炎（對照組）住院行腺樣體切除術(shù)患兒的腺樣體組織；（2）收集的標(biāo)本分為三個組：AHI＜5組、5＜AHI＜10組、AHI＞10組，分離AdMC，用ConA刺激培養(yǎng)48小時，收集上清，ELISA檢測LTC4的表達(dá)水平；（3）收集的標(biāo)本制備石蠟切片，免疫組化法檢測CysLTR1在腺樣體組織中的表達(dá)情況，免疫熒光雙標(biāo)法激光共聚焦觀察CysLTR1是否可表達(dá)于CD3+T細(xì)胞；（4）分離AdMC，分為4個組：對照組，PHA刺激組，PHA+LTD4組和PHA+LTD4+孟魯斯特納（montelukast）組，CFSE標(biāo)記法流式細(xì)胞術(shù)（FCM）檢測LTD4對PHA刺激的AdMC中CD4+、CD8+T細(xì)胞增殖的調(diào)節(jié)作用；（5）分離AdMC，分為4個組：對照組，DEX刺激組，DEX+LTD4組，DEX+LTD4組+montelukast組，Annexin Ⅴ FITC/PI雙染FCM檢測LTD4對DEX誘導(dǎo)的AdMC中CD4+、CD8+T細(xì)胞凋亡的調(diào)節(jié)作用。 結(jié)果：（1）OSAHS患兒組腺樣體CysLTR1的表達(dá)水平顯著高于對照組，CysLTR1在OSAHS患兒腺樣體組織中部分表達(dá)于CD3+T細(xì)胞，但并非優(yōu)勢表達(dá)；（2）中重度組分泌的LTC4水平顯著高于正常對照組與輕度組；（3）LTD4+PHA共同刺激AdMC后，CD4+T和CD8+T細(xì)胞的增殖率比PHA組明顯增加，montelukast可抑制LTD4的促增殖效應(yīng)；（4）LTD4+DEX共同刺激AdMC后，CD4+T和CD8+T細(xì)胞的早期凋亡率比DEX組明顯降低，montelukast可抑制LTD4的抑制凋亡效應(yīng)。 結(jié)論：（1）AdMC上清液中LTC4的表達(dá)與OSAHS患兒病情程度相關(guān)；（2）CysLTR1在腺樣體組織中表達(dá)增高，部分CysLTR1定位于CD3+T細(xì)胞，提示CysLTs可能與腺樣體組織增生肥大相關(guān)，并可能參與兒童OSAHS的發(fā)病機(jī)理；（3）LTD4對AdMC來源的CD4+T和CD8+T細(xì)胞的增殖和凋亡具有調(diào)節(jié)功能。 第三部分LTD4調(diào)節(jié)腺樣體T細(xì)胞增殖及凋亡的機(jī)制研究 目的：探討LTD4是否通過MAPKs通路調(diào)節(jié)AdMC中CD4+T細(xì)胞和CD8+T細(xì)胞細(xì)胞增殖和凋亡。 方法：（1）分離AdMC，分為對照組和處理組，處理組中加入PHA（濃度10ug/ml）和LTD4(濃度1×10-4mmol/l)，24孔板中，37℃，5%CO2孵箱培養(yǎng)。按培養(yǎng)時間點5min、15min、30min、1h、2h、12h收集細(xì)胞，提取總蛋白；（2）Western blot法檢測LTD4對MAPK通路P38、ERK1/2、JNK的磷酸化蛋白及非磷酸化總蛋白的影響；（3）分離AdMC，分為四個組：對照組、PHA組、PHA+LTD4組和PHA+LTD4+p38抑制劑SB203580組或PHA+LTD4+ERK1/2抑制PD98059組，CFSE標(biāo)記法FCM檢測，觀察CD4+T細(xì)胞和CD8+T細(xì)胞增值率的變化；（4）分離AdMC，分為四個組：對照組、DEX組、DEX+LTD4組和DEX+LTD4+p38抑制劑SB203580組或DEX+LTD4+ERK1/2抑制PD98059組，Annexin Ⅴ FITC/PI雙染FCM檢測，觀察CD4+T細(xì)胞和CD8+T早期凋亡率的變化。 結(jié)果：（1）Western Blot法檢測顯示，PHA+LTD4組中，LTD4刺激AdMC后5min后，p38磷酸化水平明顯開始增加（P＜0.05)，刺激1小時后，表達(dá)量達(dá)高峰（P＜0.001)，刺激2小時后，表達(dá)量逐漸下降，刺激12小時后，表達(dá)量降至基礎(chǔ)水平，而p38非磷酸化總蛋白的表達(dá)無明顯變化（P＞0.05)。CysLTR1抑制劑montelukast或p38的抑制劑SB203580可抑制該磷酸化反應(yīng)。（2）PHA+LTD4組中，LTD4刺激30min后，ERK1/2磷酸化水平表達(dá)水平顯著增加（P＜0.01)，刺激1小時后，表達(dá)量達(dá)高峰（P＜0.001)，刺激1.5小時后，表達(dá)量逐漸下降，刺激2小時后，表達(dá)量基本降至基礎(chǔ)水平，而ERK1/2非磷酸化總蛋白的表達(dá)在各時間點均無明顯變化（P＞0.05)。CysLTR1抑制劑montelukast或ERK1/2的抑制劑PD98059可抑制該磷酸化反應(yīng)。（3）LTD4對JNK通路的磷酸化無顯著影響。（4）CFSE標(biāo)記法FCM檢測顯示，PD98059能有效抑制LTD4調(diào)節(jié)的PHA誘導(dǎo)的ADMC中CD4+T細(xì)胞和CD8+T細(xì)胞增殖。 結(jié)論：（1）LTD4可激活A(yù)dMC中ERK1/2和p38MAPK通路，使其磷酸化水平增加，但對JNK通路無顯著影響。（2）ERK1/2信號通路可能是LTD4調(diào)節(jié)OSAHS患兒AdMC中CD4+T細(xì)胞和CD8+T細(xì)胞增殖的下游信號通路之一。（3）在OSAHS患兒AdMC中，LTD4激活了p38MAPK通路，但p38MAPK通路并未顯著影響LTD4調(diào)節(jié)增殖和凋亡的效應(yīng)，p38MAPK究竟發(fā)揮了怎樣的作用還需進(jìn)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R766

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