【作者】 高振；

【导师】 哈木拉提·吾甫尔（Halmurat·Upur）；

【作者基本信息】 新疆医科大学 ， 药理学， 2013， 博士

【摘要】 目的：选择中医药治疗COPD的常用治法,在运用系统评价和Meta分析确定中医药治疗COPD有效和安全的基础上,开展基于流行病学调查的新疆COPD和基于文献研究的内地COPD中医发病特点的对比研究,揭示新疆COPD的中医证型分布特点,找到特殊证型。在此基础上建立新疆寒燥型COPD动物模型,并从实验层面揭示寒燥型COPD的生物学基础,为COPD的方域化防治提供借鉴和参考。方法：利用系统评价和Meta分析的方法评价小青龙汤治疗COPD发作期和中医贴敷治疗COPD稳定期的疗效性和安全性；利用中医证候流行病学调查的方法揭示新疆COPD的发病特点,并将其和基于文献的内地COPD的发病特点进行对比研究,揭示新疆COPD的特殊证型。基于病因模拟-慢性应激-生物表征-药物反证的思路,利用气管滴注弹性蛋白酶结合熏烟90天建立COPD模型,在此基础上复合寒燥应激建立寒燥型COPD模型,通过肺功能、肺部病理观察和生物表征判断模型的成功建立,利用ELISA、Real-time PCR、Western-blot等方法检测模型内在病理生理状态,揭示寒燥型COPD的生物学基础。结果：1.小青龙汤联合西药内服可提高单纯西药治疗的有效率[MD=3.91,95%CI (2.50,6.12), P<0.00001],小青龙汤内服联合有创通气有效率优于单纯有创通气组[MD=3.48,95%CI (1.45,8.32), P=0.005];对于Pa02的改善小青龙汤联合西药内服优于单纯西药组[MD=7.55,95%CI (1.41,13.68), P=0.02];对于PaCO2的改善小青龙汤联合西药内服优于单纯西药组[MD=-7.11,95%CI (-9.89,-4.33),P<0.00001],小青龙汤内服联合有创通气优于单纯有创通气组[MD=-6.66,95%CI (-8.79,-4.54), P<0.00001];对于FEV1的改善小青龙汤联合西药内服优于单纯西药组[MD=6.97,95%CI (3.60,10.34), P<0.0001]。2.所有加用中医贴敷组的有效率均优于未用中医贴敷组[MD=3.63,95%CI(2.84,4.65),P<0.00001]；对于FEV1,贴敷加西药组优于西药组[MD=0.29,95%CI(0.06,0.52),P=0.01]；对于FEV1/FVC,贴敷加中药优于中药组[MD=5.29,95%CI(2.52,8.07),P=0.0002]。3.1)内地COPD总体症状中出现频次较多的是咳嗽(313,22.88%)、咳痰(204,14.91%)、气喘(124,9.06%)、喘息(120,8.77%)、气短(91,6.65%)、哮鸣音(54,3.95%)、胸闷(45,3.29%)、乏力(40,2.93%)、纳差(38,2.78%)、气促(36,2.63%)、自汗(28,2.05%)、易感冒(27,1.97%)；而新疆COPD出现频次较多的症状是气短(317次,4.43%),咳嗽(310例,4.33%),气喘(288,4.02%),咽干口干(259,3.62%),神疲乏力(244,3.41%),咳痰(235,3.28%),胸闷(227,3.17%),健忘(226,3.16%),汗出(211,2.95%),咽痒(207,2.89%),畏寒(206,2.88%),乏力(206,2.88%)；其中“燥”的表现(咽干、口干、鼻燥)为259例,占到全部调查病例的63%；“寒”的表现(恶风、畏寒、形寒怕冷、腰背冷痛、肢冷)为164例,占到全部调查病例的40%。“寒”兼“燥”的表现同时出现的为110例,占到全部调查病例的27%。内地COPD病位证素以肺(237,59.55%)、脾(81,20.35%)、肾(74,18.59%)为主,病性证素以痰(163,35.21%)、气虚(92,19.87%)、热(89,19.22%)、瘀(65,14.04%)、湿(14,3.02%)为主。证型主要以痰热蕴肺(69,19.77%)、痰瘀阻肺(43,12.32%)、肺脾气虚(33,9.46%)、肺。肾两虚(24,6.88%)、痰浊阻肺(20,5.73%)为主；新疆COPD病位证素以肺(287,61.46%),肾(142,30.41%),脾(38,8.14%)为主；病性为气虚(120,16.37%),寒(117,15.96%),阳虚(109,14.87%),痰(98,13.37%),阴虚(90,12.28%)为主。2)新疆和内地COPD患者相比舌质、舌苔和脉象总体分布不同。内地舌质瘀斑和舌质淡的多于新疆(P<0.01,P<0.05)；新疆舌质紫的则多于内地(P<0.01)；新疆舌质少津的构成比2.84%,内地舌质少津的构成比0.56%,新疆多于内地,但无统计学差异；内地患者舌质出现的胖、齿痕、绛、青则本次调查中新疆患者未见。内地黄苔和腻苔多于新疆(P<0.01),白苔和薄苔新疆多于内地(P<0.01),本次研究内地患者出现的浊苔、厚苔,本次调查新疆患者则未见。内地滑脉多于新疆(P<0.01),沉脉和弱脉多于新疆(P<0.05)；涩脉、紧脉新疆多于内地(P<0.05),浮脉和细脉新疆多于内地(P<0.01)；另外内地患者出现的无力、濡、结代、大脉本次调查新疆患者未见,迟脉则仅在新疆患者中出现,且占到7.93%。4.1) COPD组及寒燥型COPD组肺部均有肺泡断裂融合、炎症细胞浸润和小支气管下平滑肌增厚现象,肺功能寒燥型COPD组的PEF、Ti、Te、EF50均高于对照组和COPD组,COPD组的Te和EF50高于空白对照组；2)COPD组及寒燥型COPD组体质量、体质量指数和Lee’s指数均小于对照组(P<0.01),且寒燥型COPD组此三项指标均小于COPD组(P<0.01,P<0.05,P<0.01)。寒燥型COPD组每10g体质量每天进食量大于对照组和COPD组；3)寒燥型COPD组血清IL-1β含量高于空白对照组(P<0.05), IL-6、IL-8、TNF-α含量寒燥型COPD组均高于空白对照组(P<0.055、P<0.01、P<0.01), COPD组此三项指标也均高于空白对照组(P<0.01、P<0.05、P<0.05)；IL-10三组相比无统计学意义。寒燥型COPD组肺泡灌洗液IL-1p含量高于空白对照组(P<0.01), IL-6、IL-8、TNF-α含量寒燥型COPD组均高于空白对照组(P<0.01、P<0.01、 P<0.01), COPD组此三项指标也均高于空白对照组(P<0.05、P<0.05、P<0.05)；而且对于IL-6、IL-8寒燥型COPD组高于COPD组(P<0.05、P<0.05)；IL-10三组相比无统计学意义；4) MMP-2mRNA的表达量寒燥型COPD组和COPD组均高于空白对照组(P<0.01),且寒燥型COPD组高于COPD组(P<0.01)；对于MMP-9mRNA的表达量寒燥型COPD组和COPD组均高于空白对照组(P<0.01),且寒燥型COPD组高于COPD组(P<0.05)：而对于TIMP-1mRNA的表达量寒燥型COPD组和COPD组均高于空白对照组(P<0.01)；对于MMP-9mRNA/TIMP-lmRNA的比值寒燥型COPD组>COPD组>空白对照组；5)外周血中CD4+含量寒燥型COPD组低于空白对照组和COPD组(P<0.01)；CD8+含量寒燥型COPD组和COPD组均高于空白对照组(P<0.01),同时寒燥型COPD组高于COPD组(P<0.01)；CD4+/CD8比值寒燥型COPD组和COPD组均低于空白对照组(P<0.01),同时寒燥型COPD组低于COPD组(P<0.05)；6)对于AQP-5mRNA的表达量寒燥型COPD组和COPD组均低于空白对照组(P<0.01),且寒燥型COPD组低于COPD组(P<0.05)：对于MUC5ACmRNA的表达量寒燥型COPD组和COPD组均高于空白对照组(P<0.01),且寒燥型COPD组高于COPD组(P<0.01)；对于MUC5BmRNA的表达量寒燥型COPD组和COPD组均高于空白对照组(P<0.01),且寒燥型COPD组高于COPD组(P<0.05)；对于MUC5ACmRNA/MUC5BmRNA的比值COPD组<寒燥型COPD组<空白对照组；7)对于AQP-4、AQP-5蛋白的表达量寒燥型COPD组低于COPD组,COPD组低于空白对照组；对于MUC5AC、MUC5B的表达量寒燥型COPD组高于COPD组,COPD组高于空白对照组；8)大鼠模型肺部IL-8与TIMP-1、MMP-2、MUC5B相关,IL-8与MMP-9、MUC5AC密切相关,IL-8与AQP5负相关；IL-10与MMP-2、MMP-9负相关,IL-10与AQP5相关；TNF-a与MUC5AC密切相关；IL-6与TIMP-1相关,IL-6与AQP5负相关,IL-6与MMP-2、MMP-9、MUC5AC、MUC5B密切相关；TIMP-1与MMP-2、MMP-9相关；MMP-2与MMP-9、MUC5AC、MUC5B密切相关,MMP-2与AQP-5负相关；MMP-9与MUC5AC、MUC5B密切相关,MMP-9与AQP-5负相关；AQP5与MUC5AC、MUC5B负相关；MUC5AC与MUC5B密切相关。结论：1.发现中医内、外疗法治疗COPD均可显示出一定的疗效,但针对性不同,可与西医治疗实现优势互补。小青龙汤内服联合西医治疗COPD,可一定程度提高临床疗效,具体表现在增加有效率,提升Pa02、降低PaCO2上,同时可以某种程度提高患者FEVI。中医贴敷作为外治法如果联合内服药,则应根据患者表现选择相应的西药或中药联合。中医贴敷可以提高西药对FVC和FEV1改善的疗效,而中医贴敷不能提高中药对FEV1的改善程度。中医贴敷可提高中药对FEV1/FVC的改善,但对西药反而没有协同作用。2.指出寒燥诱导和加剧了COPD的发病,是新疆COPD异于内地COPD发病、方域化发病的重要病因病机特点之一。体现在证型上则寒燥型是新疆COPD发病的特殊证型和多发证型之一,体现在病机上则该证型“局部燥,全身寒”、“内燥外寒”,体现在程度上则寒燥型属COPD的一个早期证型。内地COPD发病主要是痰、热、瘀为标,肺、脾、肾虚为本,痰、瘀、虚贯穿COPD发作期和缓解期的各个环节,是病机的主要环节所在,症状表现以咳嗽、咳痰、气喘、气短为主,临床治疗中应该注意祛痰、活血、补气药物的应用。而新疆COPD的发病则以气虚、寒、阳虚为主,肺、肾、脾虚为本,症状表现以气短、咳嗽、气喘、咽干口干、神疲乏力为主。临床治疗中应该注意祛痰止咳、温肺润燥、补气药的应用。3.证实熏烟结合气道滴注弹性蛋白酶90天可以成功复制COPD模型,而在此基础上施加寒燥环境刺激建立新疆寒燥型COPD模型是可行的。寒燥型COPD模型表现为体型消瘦、体重下降、毛色黄无泽,气道内可闻及痰鸣音,体质量指数和Lee’s指数减小,饮水、进食量增加且食物利用率低；PEF和EF50值降低、Ti、Te值增高。具有温肺润燥功效的中药止嗽散加减方对改善该病证的症状及肺功能有明显效果。但鉴于本证病机,法当辛开温润,不可过用温热而助燥伤津。4.揭示寒燥通过COPD肺部AQP5mRNA和MUC5ACmRNA、MUC5BmRNA表达及其相应蛋白分泌的调节,降低了水通道蛋白的分泌,增加了黏蛋白的分泌,打破了水通道蛋白和黏蛋白的平衡,进而影响了气道黏液的功能状态,增加气道阻塞程度,促进以肺部炎症反应为主的肺部及全身炎症反应,扰乱了机体的免疫功能,导致机体肺气虚、卫外功能减弱,加重COPD的蛋白酶-抗蛋白酶失衡,进而促进和加重了COPD发病,可能是寒燥型COPD的重要生物学基础。更多还原

【Abstract】 Objective:To select common Chinese traditional medicine treatment for COPD. On the basis of effective and safe therapy on COPD, we use systematic review and meta-analysis to observe the characteristics between COPD patients in Xinjiang province based on epidemiological investigation and COPD patients in other provinces based on literatures. To observe the TCM syndrome distribution characteristics of COPD in Xinjiang and find the special pattern of syndrome. On this basis, to establish Xinjiang cold-dryness animal models of COPD. To provide evidences for revealing the biological mechanism of the cold-dryness COPD and regionalized prevention and treatment for COPD.Method:To evaluate the effect and safety of Xiaoqinglongtang for treatment of COPD and TCM sticking therapy for patients with stable COPD by using systematic reviews and meta-analysis. To reveal the characteristics of COPD in Xinjiang by using TCM syndrome epidemiological investigation, then to compare characteristics of COPD between them in Xinjiang and them in other provinces based on reports in literatures. Based on the method of etiologic simulation-chronic stress-biological phenotype-pharmacal disproof, to establish the COPD models by instilling elastinase into trachea and fumigating for90days. We determined the success of model construction by observing pulmonary function, pathological change and biological phenotypes. To detect the pathophysiological change by Elisa, real-time PCR and Western-blot.Results:1. the effective rate of combing western medicine and Xiaoqinglongtang is higher than only using western medicine [MD=3.91,95%CI (2.50,6.12), P<0.00001]. The effective rate in group of Xiaoqinglongtang combined with invasive ventilation is better than simply invasive ventilation group[MD=3.48,95%CI (1.45,8.32), P=0.005]. For the improvement of PaO2western medicine combined with Xiaoqinglongtang is superior to simple western medicine[MD=7.55,95%CI (1.41,13.68), P=0.02]; For the improvement of PaCO2western medicine combined with Xiaoqinglongtang [MD=-7.11,95%CI (-9.89,-4.33), P<0.00001] and Xiaoqinglongtang combined with invasive ventilation [MD=-6.66,95%CI (-8.79,-4.54), P<0.00001] are all superior to simple western medicine. For the improvement of FEV1, western medicine combined with Xiaoqinglongtang is superior to simple western medicine [MD=6.97,95%CI (3.60,10.34), P<0.0001].2. The effective rates of COPD in the groups combined with external application are superior to the groups without external application[MD=3.63,95%CI (2.84,4.65), P<0.00001]; For improvement of FEV1, the group of western medicine combined with external application is superior to simple western medicine group. For improvement of FEV1/FVC, the group of TCM combined with external application is superior to simple TCM group [MD=5.29,95%CI (2.52,8.07),P=0.0002].3.1) The most frequent symptom of COPD in other provinces are cough (313,22.88%), sputum (204,14.91%), asthma (124,9.06%) respite (120,8.77%), shortness of breath (91,6.65%), wheeze (54,3.95%), chest tightness (45,3.29%), fatigue (40,2.93%), anorexia (38,2.78%), shortness of breath (36,2.63%), spontaneous (28,2.05%), colds (27,1.97%); The most frequent symptom of COPD in Xinjiang are shortness of breath (317,4.43%), cough (310cases,4.33%), asthma (288,4.02%) throat, dry mouth (259,3.62%), lassitude (244,3.41%), sputum (235,3.28%), chest tightness (227,3.17%), forgetfulness (226,3.16%), sweating (211,2.95%), itchy throat (207,2.89%), chills (206,2.88%), fatigue (206,2.88%). The patients with the clinical manifestations "dry"(dryness of the mouth and throat, dry nose) are259cases, accounting for63%of in total cases under investigation; patients with the " cold " manifestations (evil wind chills, shaped cold, cold back, limbs) are164cases, accounted for40%of all investigated cases., patients with both symptoms are110cases, accounting for27%of all cases under investigation. The COPD symptoms in other provinces are slip known for lung (237,59.55%), spleen (81,20.35%), kidney (74,18.59%), disease nature elements sputum (163,35.21%), Qixu (92,19.87%), fever (89,19.22%), stasis (65,14.04%), wet (14,3.02%) the main. Syndrome type phlegm retention in the lung (69,19.77%), phlegm lung (43,12.32%), DLS (33,9.46%), lung and kidney deficiency (24,6.88%), phlegm blocking lungs (20,5.73%); The symptoms of COPD in Xinjiang are Slip known for lung (287,61.46%), kidney (142,30.41%), spleen (38,8.14%); disease deficiency (120,16.37%), cold (117,15.96%), Yangxu (109,14.87%), sputum (98,13.37%), Yinxu (90,12.28%).2) Tongue with cchymosis and pale in mainland was higher than in Xinjiang (P<0.01, P<0.05); tongue with purple in Xinjiang was higher than in mainland (P<0.01); constituent ratio of less of body fluid was2.84%in Xinjiang, but in mainland was0.56%, but made no difference; fat tongue, scalloped, purple, green tongue appearance in mainland but not in Xinjiang, yellow fur and greasy fur in mainland was higher than in Xinjiang (P<0.01); and white and thin fur in Xinjiang was high than in mainland (P<0.01); moss, thick fur appearance in mainland but not in Xinjiang, slippery pulse in mainland was higher than in Xinjiang (P<0.01); pulse astringent, tight pulse in Xinjiang were higher than in mainland (P<0.05); floating pulse and veinlets pulse in Xinjiang was higher than in mainland (P<0.01); weakness, soft pulse, intermittent pulse, large pulse just appearance in mailland; slow pulse will only occur in patients in Xinjiang, and accounted for7.93%.4.1) Rats in COPD group, and cold-dry COPD group, we can observed the alveolar fracture fusion, infiltration of inflammatory cells and bronchial smooth muscle thickening, the lung function in cold-dryness COPD group, PEF, Ti, Te, EF50, were higher than them in COPD group, Te and EF50in COPD group were higher than them in control group;2) the body weight, body mass index, and Lee’s index in COPD group and the cold-dryness COPD group were lower than them in control group (P<0.01), and these three parameters in cold-dryness COPD group were lower than them in COPD group (P<0.01, P<0.05, P <0.01). The daily food consumption per lOg body weight in cold-dryness COPD group was greater than it in control group and COPD group;3) Serum IL-1beta in cold-dryness COPD group was higher than that in control group (P<0.05), Serum IL-6, IL-8, TNF-alpha in cold-dryness COPD group were higher than them in control group (P<0.05, P<0.01, P<0.01), and these three parameters in COPD group were also higher than them in control group (P<0.01, P<0.05, P<0.05); the level of IL-10among three groups was not statistically different. The cold-dryness COPD BALF IL-lbeta content was higher than them in control group (P<0.01), IL-6, IL-8, TNF-alpha content in cold-dryness COPD group were higher than them in control group (P<0.01, P<0.01, P<0.01), and they were also higher in COPD group than them in the control group (P<0.05, P<0.05, P<0.05); and IL-6, IL-8was higher in cold-dryness COPD group than them in the COPD group (P <0.05, P<0.05); IL-10among three groups was not statistically different.4) MMP-2 mRNA expression level in cold-dryness COPD and COPD groups were higher than them in control group (P<0.01), and it was higher in cold-dryness COPD group than in the COPD group (P<0.01); MMP-9mRNA expression in both COPD and cold-dryness COPD groups were higher than it in control group (P<0.01), and it was higher in the cold-dryness COPD group than in the COPD group (P<0.05); TIMP-1mRNA expression level of cold dry COPD group and COPD group were higher than it control group (P<0.01); the ratio of MMP-9mRNA/TIMP-1mRNA was highest in cold-dryness COPD group (cold-dryness COPD group>COPD group>control group);5) peripheral blood CD4+in cold-dryness COPD group was lower than it in control group and COPD group (P <0.01); CD8+in cold-drynes COPD and COPD groups were higher than it in control group (P<0.01), while it was higher in cold-dryness COPD group than COPD group (P<0.01); ratio of CD4+/CD8+were lower in cold-dryness COPD and COPD groups (P<0.01), while the it was also lower in cold dry COPD group than in COPD group (P<0.05);6) The AQP-5mRNA of the expression in cold-dryness COPD and COPD groups were lower than it in control group (P<0.01), and it was lower in cold-dryness COPD group than it in the COPD group (P<0.05); MUC5AC expression in cold-dryness COPD and COPD groups were higher than it in control group (P<0.01), and it was higher in cold-dryness COPD group than it in the COPD group (P<0.01); MUC5BmRNA expression in cold-dryness COPD and COPD groups was higher than that in control group (P<0.01), and the cold dry COPD group was higher than the COPD group (P<0.05); ratio of MUC5ACmRNA/MUC5BmRNA was lowest in COPD group (COPD group<the cold dryness COPD group<control group).7) The expression of AQP-4, AQP-5protein in cold dry COPD group were lower than in the COPD group, COPD group was lower than control group; the expression of MUC5AC, MUC5B were highest in cold-dryness COPD group;8) IL-8in lung of rats is closely related to TIMP-1, MMP-2, MUC5B-related the IL-8and MMP-9, MUC5AC. IL-8was negative related to AQP5. IL-10was associated with the expression of MMP-2, MMP-9. IL-10and AQP5were closely negative related to TNF-alpha and MUC5AC; IL-6and TIMP-1, IL-6and AQP5; IL-6and MMP-2, MMP-9, MUC5AC, MUC5B were closely related to TIMP-1and MMP-2; MMP-9; MMP-2and MMP-9, MUC5AC, MUC5B were closely negative related to MMP-2and AQP-5; MMP-9and of MUC5AC, MUC5B were closely related to, MMP-9was negative related to AQP-5; AQP5was negative related to MUC5AC and MUC5B; MUC5AC was correlate with MUC5B. Conclusion:1. We found that the Chinese internal medicine and external application treatment of COPD were effective for COPD, but for different targets, it was complementary advantages combined with Western medicine treatment. Xiaoqinglongtang combined with Western medicine treatment of COPD may, to some extent, improve the clinical efficacy, specifically manifested in increased efficiency and improve PaO2, lower PaCO2, and it also can improve patients’ FEV1. TCM sticking as external treatment combined with medicine should be based on the symptoms of patient. TCM sticking combined with Western medicine can improve the FVC and FEV1. However we did not observe th improvement of FEV1when combined the traditional Chinese medicine with Chinese medicine. TCM sticking combined with TCM improved FEV1/FVC, but there is no synergy with Western medicine.2. We determined that the cold the dry induced and increased the incidence of COPD, which is the one of different characteristics from the COPD in other provinces. When concerned the pattern of syndrome,"cold-dryness" is the special permit and prevalent type of COPD in Xinjiang. When concerned the pathogenesis of the syndrome type, It characterized by " local dry, the whole body cold "and "within the dry, cold outside", when concerned the degree of COPD, cold-dryness is an early COPD syndrome type. Characteristics of COPD in othere provinces were heat, stasis as the phenotypes, deficiency of lung, spleen, kidney is the base; phlegm, blood stasis, virtual throughout the COPD exacerbation and remission of all aspects of the pathogenesis, the major symptoms are cough, sputum, wheezing, shortness of breath, phlegm, blood, qixu. Medicine application in clinical treatment should pay attention to those. Characteristics of COPD in Xinjiang are sputum, cloud, cold, dry as main phenotypes, and deficiency of lung, kidney, spleen as basis, shortness of breath, coughing, wheezing, dry throat, dry mouth, lassitude as main symptoms. Doctors should pay attention to cough expectorant, warm the lung dryness, drug application for qixu.in the clinical treatment of COPD.3. We confirmed that fumes combined with Aikido infusion of elastase for90days can successfully construct the COPD model, and on the basis of which, cold-dry environ-ment can stimulate and establish Xinjiang cold-dryness COPD model. cold-dryness COPD models have some characters such as body weight loss, weight loss, disluster hair color, sputum in airway, decreases in body mass index and Lee’s index, water and food consummation increased, but food utilization is lower; PEF and EF50reduced, Ti and Te value increased. Warm the lung moistening effect of traditional Chinese medicine Cough Powder plus or minus side to improve the certificates of disease symptoms and lung function had a marked effect. But in view of the pathogenesis of the card, when Acrid moist, not too warm and dry to help disability allowance4. To reveal cold dry through the COPD lungs AQP5mRNA and MUC5AC, MUC5BmRNA expression and its corresponding regulation of protein secretion, reducing the water channel protein secretion, increasing mucin secretion, breaking the balance of aquaporin and mucin, thereby all these affect the functional status of the airway mucus, increase the degree of airway obstruction, promote the main pulmonary inflammatory response of the lungs and systemic inflammatory response, disrupt the body’s immune function to cause lung qixu of the body, protection of the lung weakened, aggravate COPD protease-anti-protease imbalance, thus contributing to increased incidence of COPD, may be an important biological basis of cold-dryness COPD.更多还原