【作者】 黄仕营；

【导师】 钟嘉熙；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2010， 博士

【摘要】 背景：发现登革热迄今至少有200多年的历史,进入20世纪,登革热在世界范围内的流行规模不断扩大,流行频度不断增加。登革热一度在我国绝迹30余年,1978年首次在佛山市报道登革热暴发疫情,继而在东南沿海频频流行。其中广东省是登革热流行的重灾区,而全省的63%发生在广州。随着广州城市化的进程,登革热流行有上升的趋势,控制效果如何,直接影响一个城市的经济和形象。如何有效防治登革热,成为亟待解决的公共卫生课题。登革热西医治疗目前尚无特效方法,主要采用对症、支持疗法。中医药治疗(尤其是岭南温病指导下)登革热有独特的疗效,如何对中医药疗法进一步研究,合适的动物模型是关键。目的：通过文献综述、临床病例回顾性研究,了解中医药治疗登革热的概况,以及既往模型研究的薄弱环节,借以寻求启发：通过不同鼠类、不同热暴露方式和不同攻毒模式等复合因素来造模,筛选较为理想的登革热湿热证动物模型；在此基础上,验证清热祛湿法的治疗效果及作用靶点,研究湿热证与水通道蛋白的关系。方法：1、文献研究：采用文献综述的形式,整理出中医药对登革热的认识和诊疗概况,以及登革热鼠类模型的研究进展,分析岭南登革热的湿热特征,注重其对本次实验和临床研究的启发作用。2、实验研究：分模型建立和评价,以及清热祛湿法干预作用探讨两部分。①、登革热湿热证模型的建立：本实验动物分4周龄左右的BALB／c和C57BL／6小鼠,分热习服和短暂热暴露,以及分作单次登革热病毒2感染(简称DF2)和ADE效应(简称ADE)两种不同的攻毒模式；按照排列组合原则,得8个造模组,同时设立空白对照组、单纯DF2对照组、湿热条件对照组,共得14组。观察实验小鼠体温、症状、体征,以及检测血小板(PLT)、肝功能(ALT、AST)、血脂(TG、CHOL)和肝脏病理等,来评价造模效果,筛选出最佳登革热湿热证动物模型；②、不同药物对登革热模型小鼠干预作用比较及机理探讨：选用实验一筛选出的动物随机分为5组：模型组、蒿芩清胆汤治疗组、清瘟败毒饮治疗组、病毒唑治疗组、生理盐水治疗组,再设立正常对照组,观察各组实验小鼠体温、症状、体征,检测血小板(PLT)、肝功能(ALT、AST)、血脂(TG、CHOL)和肝脏病理,以及脑水通道蛋白4(AQP4)、肾水通道蛋白2(AQP4)、胃水通道蛋白4(AQP4)；比较各种治疗药物的治疗作用,探讨不同治法的治疗机理。3、临床研究：回顾研究2006年451个广州市第八人民医院住院病例；按照统一标准分为湿热疫和暑燥疫,观察两组患者年龄分布,比较其平均年龄、性别、体温峰值、皮疹发病率、肝功能损害(ALT、AST升高)情况、血小板水平等；再区分两组患者中,结合中医辨证施治者和单纯西医治疗者,比较其平均退热时间、皮疹消退时间、血小板复常时间、平均病程及ALT、AST水平的影响,以判断其治疗效果。结果：1文献研究：①、岭南温病的湿热特征对登革热具有一定影响；②、中医药治疗登革热疗效优于单纯西医治疗；③、登革热模型不够理想,有诸多原因,思维方面没有突破可能是重要的原因之一：④、“病证结合”思想的应用在登革热湿热证动物造模方面的应用大有前途。2、实验研究：①、一般表现：小鼠病毒攻击后出现疲倦状态,体温稍降低,活动明显减少,对刺激反应慢,毛发耸立不顺,欠光泽,饮食、饮水量减少,小便较臭、大便较正常组软；进入气候仓后表现出现烦躁不安,活动增加,食量减少,饮水增多,渐渐至倦卧,对刺激反应慢,多次放入气候仓后形成热习服,则与正常小鼠无明显差异。BALB/c和C57BL／6小鼠之间相差不大。蒿芩清胆汤对改善造模组的症状有一定作用；②、体温检测：各组小鼠病毒攻击后出现体温下降,然后逐渐恢复正常；短暂热暴露可造成体温升高,热习服后则升高不明显,各组间存在差异。蒿芩清胆汤及清瘟败毒饮有降低体温的作用；③、主要指标的检测：小鼠病毒攻击后,PLT变化情况相似,均有不同程度的降低；小鼠的ALT及AST较正常组有不同程度升高,尤其是AST变化显著；给予高脂饲料饲养的小鼠TG、CHOL均有所升高；但热习服组肝功能及血脂升高不明显。蒿芩清胆汤有降低ALT、AST以及TG、CHOL的作用,清瘟败毒饮有对降低的PLT有提升作用；④、复合因素造模后,模型组脑组织AQP4的表达增强,胃粘膜AQP2的表达增强；而肾内AQP2的表达减弱；蒿芩清胆汤有逆转上述表达的作用,使之回调,与其余各组相比,差异有统计学意义；并对以上三个AQP进行相关分析,自变量肾AQP2对因变量胃AQP4有显著相关；自变量脑AQP4对因变量胃AQP4无相关关系；⑤、肝脏病理变化：各造模组均有标本呈不同程度病理变化：肝细胞轻到重度水肿；个别细胞嗜酸性变；肝细胞点状、小灶状坏死,坏死区较多炎细胞浸润；未见脂肪变,未见纤维组织增生。湿热条件干预组小鼠肝脏镜检可见见肝细胞轻度肿胀；蒿芩清胆汤治疗后肝细胞状态有所好转。3、临床研究：①、湿热疫与暑燥疫患者的年龄分布各有特点,但在年龄、性别、并发肝功能损害情况等方面无差别,而暑燥疫患者在出现高热、合并出现皮疹及血小板降低等方面明显高于湿热疫组；②、两个证型患者中,中西医结合治疗组在平均退热天数、皮疹消退天数、平均住院病程均少于单纯西医治疗组,暑燥疫患者又少于湿热疫患者；③、在血小板复常时间方面,湿热疫组与对照组无显著性差异,而暑燥疫组少于单纯西医治疗对照组和湿热疫组；④、两组患者给予中西医结合治疗后,7天内及7～14天内,其肝功能水平与单纯西药治疗对照组比较无差异。结论：1、实验研究结论：综合症状、体温、肝功能损害以及血脂多方面衡量各个模型组,以BALB/c+DV2+热损伤组造模效果,最能体现登革热湿热证的特点；蒿芩清胆汤对以上的登革病毒湿热证模型有治疗作用,能改善症状,下调血脂,保护肝功能改善肝脏病理损害,调整失常的水通道蛋白；2、临床研究结论：登革热暑燥疫合并高热、出现皮疹及血小板降低的情况明显多于湿热疫,中西医结合治疗登革热在退热、消除皮疹和升高血小板方面的疗效明显优于单纯西药对症治疗,暑燥疫组又优于湿热疫组,但在改善肝功能方面二者尚无差别。更多还原

【Abstract】 Backgroud:Dengue fever popular at least had more than 200 years until now. The popular scale and frequency of DF have expanded endlessly since 20 cen-turies. DF once vanished more than 30 years in our country, Foshan reported Dengue fever epidemic situation for the first time in 1978, and then repeated in the popular southeast coast citys frequently. Among them, Guangdong Province is the hardest hit by dengue fever, and 63% events of the province occurred in Guangzhou. With the urbanization of Guangzhou, DF has a rising trend, how to effective control will affect it’s economy and image directly. Effective prevention and controltion of DF, becomes a public health issue to be resolved hastily.There are no special effective methods for DF’s treatment current-ly, mainly about symptomatic and supportive therapy. Treatments of TCM on DF has a unique effect, how to further study Chinese medicine, a suitable animal model will play a key pole.Objective:According to literature review and retrospective clinical Case Study, we hope that we will understand the profile of Chinese medicine treatment of dengue fever, as well as the weak link in previous models, and then to seek enlightenments on the study; of different rodents, different methods and different heat exposure attacking multiple factors such as drug models modeling of the effects in order to find an ideal animal model for dengue Damp; On this basis, verify the effect of heat treatment of dampness and the function of target, of damp and water channel protein.Methods:1、Literature:to finish a Chinese medicine clinic on the awareness and profile of dengue fever and dengue fever murine model of progress by literature reviews and analysis the damp-heat characteristic of dengue in Lingnan, which will be focuse on it’s inspiring role in this experiment and clinical research. 2、Experimental Studies:there are two parts which were the model building and evaluation and the intervention effects of clearing heat and dampness.①、To establish Damp-heat model of dengue fever:The 4 weeks old BALB /c and C57BL/6 mice were divided into short-term heat acclimation and heat exposure, while divided into single dengue virus 2 infections (referred to as DF2) and ADE effects (the ADE) model of two different virus challenge respectively;So, in accordance with the principles of permutations and combinations, there were 8 model. Finally, there were 14 group adding the blank control group, DF2 control group, damp-heat group included 14 group. Then through the observation of mice body temperature, symptoms, signs, and platelet (PLT), liver function (ALT, AST), blood lipids (TG, CHOL) and liver pathology, to evaluate the results of model establishment and filter out the best damp-heat syndrome of dengue fever in animal;②、To compare and investigate the mechanism of the intervention effects of different drugs on mice of Damp-heat model of dengue fever: Mice selected by the first experiment were divided into 5 groups randomly: normal group, model group, Artemisia Scutellaria Decoction treatment group, treatment group Qingwenbaiduyin drink, Ribavirin treatment group, saline treated group. Then to observe body temperature, symptoms, signs, platelet (PLT), liver function A (LT, AST), blood lipids (TG, CHOL) and liver pathology, and to detect brain aquaporin 4 (AQP4), renal water channel protein 2 (AQP2), stomach aquaporin 4 (AQP4). Finally, to compare the therapeutic effects of different drug treatments and explore their therapeutic mechanism.3、Clinical Research:Review of 451 cases in-hospital in Guangzhou 8th People’s Hospital in 2006; All cases were divided into damp-heat group and summer-dry group in accordance with certain standards, then to compare the age distribution, average age, gender, body temperature peak, the incidence of rash and impaired liver function (ALT, AST increase), plate-let levels, etc. Further to divided those 451 cases into two groups:group with combinative treatment and group with western medicine and compare the mean fever clearance time, rashes subside time, normalization of platelet time, the average duration of illness and ALT, AST levels, to determine its efficacy.Results:1、Literature Review:①The damp-heat factors of seasonal febrile disease in Lingnan had a certain effect on dengue fever.②Chinese medicine threpy in dengue fever had more effective than western medicine.③Animal model of dengue fever wasn’t ideal. There were many reasons possibly, but it’s likely to be the most important reason to have no breakthrough in thinking;④The combination of disease and syndrome maybe have a promising future. 2、Experimental Research:①、General Performance:After a virus attack, mice indicated fatigue condition, lower temperament, less movement, tired lying, slow respond to stimuli, hair standing on flow, less shiny, less diet and water intake, more smelly urine and soft feces. Into the warehouse, mice manifested irritability, less activities, less diet and respond to stimuli, more water intake and tired lying. After put into the warehouse several times. The difference of BALB/c and C57BL/6 mice wasn’t significant. Haoqin-qingdan decoction had paid a certain role in improving the symptoms of model group.②、Body Temperature Test:After the virus attacks, the temperatures of mice slowed down, and then gradually returned to normal; short-term heat ex-posure could cause fever increasing, but became minute after heat ac-climation. The differences among the groups are significant. Both Hao-qinqingdan decoction and Qingwenbaiduyin decoction had the effects of decreasing body temperature.③、Detection of the main indexes:After virus attacks, PLT slowed down to a certain extent similarly. ALT and AST increased to varying degrees, especially AST;TG, CHOL increased in mice given high fat diet fed. But liver function and blood lipids had no significant increase in heat ac-climation group. So Haoqin decoction could decrease ALT, AST and TG, CHOL, while Qingwenbaidu decoction could upgrade PLT.④、After established modle with complex method, the expression of AQP4 in brain tissue and AQP2 in gastric mucosa increased, but renal AQP2 re- duced. Haoqin decoction could reverse those expressions to the nomal levels, and the differences were statistically significant, compared with other groups. Through correlation analysis three AQP above, the in-dependent variables of renal AQP2 had a significant correlation with the dependent variable of gastric AQP4 and The independent variables of renal AQP2 had no correlation with the dependent variable of gastric AQP4.⑤、Liver pathology:There are varying pathological changes in specimens of each groups. Liver cells had edema from mild to sever. Individual cells had eosinophilic changes;Liver cells had punctate, small focal necrosis, and necrosis area was infiltrate by inflammatory cells; There was no steatosis and fibrosis. Microscopy shows slight swelling of liver cells in group with damp-heat intervention; The state of liver cells had im-proved after the treatment of Haoqinqingdan decoction.3、Clinical Studies:①、Patients with dry-summer plague and damp-heat plague had dif-ferent characteristics in age distributionh, but no dif- ference in age, sex, concurrent liver damage. Summer-dry group were signi-ficantly higher than damp-heat group in the event of epidemic fever, rash and platelet decrease; ②、The integrated therapy group were less than the western medicine group in the average number of days fever, rash subsided days, the average duration of hospital stay, and summer-dry group were less than damp-heat group in those indexes;③、In platelet recovery time, the difference between the group with damp-heat plague and control group wasn’t significant, but the group with summer-dry was less than the group with western therapy and control group and the difference was significant;④、After 7 days or 7-14 days treatment with integrative medicine, there was no difference between two groups with integrative medicine and the group with western medicine in liver function.Conclusion:1、Experimental Studies Conclusion:According with symptoms, fever, liver damage and blood lipid, the model group of BALB/c+DV2+thermal indicated damp-heat characterristic of dengue fever, so it’s the best method. Haoqin decoction had therapeutic effects on mice with damp-heat syndrome of dengue fever, while have improved symptoms, decreased blood lipids and liver pathological damage, and regulated the water channel protein adjustment disorders;2、Clinical Research Findings:the dry-summer plague had more severe fever, skin rash and lower platelet than the damp-heat plague. Integrated Treatment had more significant effects than western medicine treatment in improvement those symptoms, in which the summer-dry group was better than damp-heat group, but the difference of improvement liver function wasn’t significant.更多还原