【作者】 王玲；

【导师】 周铭心；

【作者基本信息】 新疆医科大学 ， 药理学， 2015， 博士

【摘要】 目的:设计制作西北燥证动物模型,以宏观辨证指标及经验方药反证评价模型,同时,对模型动物以部分现代医学指标进行检测与分析,使得西北燥证及其相关性疾病的现代医学机理与药物防治研究深入至微观层次。方法:1)系统总结自西北燥证概念提出至今开展的研究工作,从中医学的整体观念出发,分析西北燥证的多元病因,梳理西北燥证的复杂性证候结构,从而揭示其病机本质,确立西北燥证的基本治法和治疗方药,为西北燥证证候模型研究打下理论基础;2)依据西北燥证病因、症状及目前常用实验动物的生物学特点,选择适宜西北燥证动物模型研究用的动物品系;3)借鉴既往文献六淫动物造模经验,以西北燥证病因研究结果为依据,利用其燥火风寒气化与实际气候指标间因子负荷关系,参照从西北燥证易发地区和田之实际气候所统计的燥火风寒气化构成比,选定实验室人工气候指标并加以分级、组合、量化,以时间与空间相结合变换调控人工气候指标因素,订定实验方案;4)分析人体四诊和动物表征的对应关系,从西北燥证人体辨证（计量学辨证）向动物辨证过渡,建立西北燥证动物计量辨证方法;5)将实验动物随机分为正常组、模型组和方药治疗组进行8个气化周期（1个周期为6d）的病因模拟造模;6)以西北燥证动物计量辨证量表为主要评价模型指标,选择饮水、饮食量、大便性状、体重变化结合方药反证评价动物模型,并确立造模所需气化周期数;7)光学显微镜下观察模型动物肺、颌下腺组织,透射电镜观察模型动物肺、颌下腺组织的细胞内结构,评价代表性器官在造模因素作用下的损伤程度;8)用流式细胞术检测外周血T淋巴细胞在造模过程中的变化,说明造模期间T淋巴细胞亚群的动态变化。ELISA法检测实验动物外周血白介素1-β（IL-1β）、雌二醇（E₂）、睾酮（T）的含量,评价造模因素对实验动物免疫能力及性激素水平的影响;9)以TUNEL法检测实验动物代表性器官肺及颌下腺组织细胞的凋亡状况,评价造模因素对实验动物的损伤程度;10)以免疫组织化学法检测实验动物肺及颌下腺凋亡相关蛋白bcl-2、Bax、fas-l的表达,初步探索细胞凋亡的可能途径;11)ELISA法检测实验动物外周血促肾上腺激素（ACTH）、皮质醇（Cortisol）、儿茶酚胺（CA）的含量,评价造模因素对神经内分泌免疫网络HPA轴的作用;12)采用TBA法测定血清中丙二醛（MDA）浓度及黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）活力,评价造模因素;13)用流式细胞术检测外周血T淋巴细胞在造模过程中给予经验方药治疗的变化,说明经验方药对模型动物T淋巴细胞亚群作用的动态变化;14)以“燥、火、风、寒”气化柜（即西北燥证主证造模因素）联合灌胃甲状腺素的方法建立西北燥证兼心肾阴虚动物模型,流式细胞术检测外周血T淋巴细胞经时变化,并以析因设计考察两造模因素对fT3、fT4的影响;15)“燥、火、风、寒”气化柜联合卵蛋白致敏法制作西北燥证兼支气管哮喘模型,ELISA法检测实验动物血清IgE、IL-4、IFN-γ水平,评价两因素及经验方药的作用。结果:1)课题组以往研究揭示西北燥证是外因于燥、火、风、寒合邪,内因于燥敏体质状态,而出现内、外燥证兼发的繁杂证候;据辩证论治原则筛选出西北燥证主证西北燥证兼心肾阴虚证及西北燥证兼支气管哮喘的经验方药;2)选定温度、湿度、光照、风吹、沙尘等5项人工气候指标为造模因素,并据以改装人工气候箱、加设沙尘实验箱,同时为5项人工气候指标分级量化赋值并加以组合以体现燥火风寒气化属性,拟定气化周期状态公式,制定人工气候指标因素时空变换调控计划,优选SD大鼠进行西北燥证主证动物模型造模;3)与对照组相比,模型组大鼠在造模因素施加后即出现饮水量增加的现象,并持续直至处理结束。在处理的前3个气化周期饮食量减少明显,但随着处理的施加,至第8d开始,模型组的饮食量与正常对照组相比无统计学差异。与对照组相比,模型组大鼠的体重在施加处理的第4⁶d开始体重增长缓慢,P=0.001（P<0.05）差异具有统计学意义,持续至10¹2d;第13d及以后模型组与对照组体重比较差异均不具有统计学意义。与对照组相比,模型组大鼠在造模因素施加期间始终有便秘现象的出现;4)病理组织切片HE染色提示,24d及之前模型的肺及颌下腺组织未见明显病理改变,48d组个别动物开始出现较明显的病理损伤;5)外周血细胞计数结果显示,各组实验动物血细胞计数均在正常值范围内,与正常对照组相比,24d时,单核细胞比率（Mon%）升高、嗜酸性粒细胞百分比（Eos%）降低具有统计学意义;6)从主证动物模型外周血T淋巴细胞经时变化可见,与正常对照组相比:CD3+、CD4+、CD8+细胞百分含量随时间变化,但差异不具有统计学意义,CD4+/CD8+比值随时间下降、6d时差异具有统计学意义（P=0.019）,表明造模因素可降低CD4+/CD8+比值。ELISA检测实验动物外周血血清IL-1β、E₂、T含量,与正常对照组相比,各时间段处理组IL-1β、T水平的降低均具有统计学意义,而E₂水平的降低不具有统计学意义;7)透射电镜下观察实验动物肺及颌下腺组织细胞内结构,模型组出现明显凋亡现象、细胞膜损伤、细胞肿胀、肺泡间可见嗜酸性粒细胞浸润等现象;8)TUNEL实验结果可见模型组肺泡、支气管周围及颌下腺浆液性细胞的细胞核有明显黄色至褐色染色的凋亡细胞;9)免疫组织化学法检测实验动物肺及颌下腺凋亡相关蛋白的表达可见:模型组bcl-2表达不明显、bax、fas-l有较多表达,说明造模因素可能造成促凋亡蛋白的表达上调;10)ELISA检测实验动物外周血血清ACTH、Cortisol及CA含量,与正常对照组相比,模型大鼠血清内促肾上腺皮质激素（ACTH）含量无差异,而皮质醇（Cortisol）含量有所下降（P=0.020）,儿茶酚胺（CA）含量也有所降低（P=0.019）。与模型组相比,低、中、高剂量的给药组能提高血清儿茶酚胺（CA）的含量（P=0.028,P=0.000,P=0.000）;同时低、中、高剂量的给药组能提高皮质醇（Cortisol）含量恢复至正常水平,与模型组的差异具有统计学意义（P=0.007,P=0.000,P=0.000）;中、高剂量的给药能提高ACTH的含量,与模型组相比具有统计学意义（P=0.034,P=0.006）;11)与正常对照组相比,模型组大鼠血清IgE水平升高,给与低剂量和中剂量方药后降低至与正常对照组无差别,高剂量给药组大鼠血清IgE未见降低,与正常对照组相比水平较高,具有统计学意义。与正常对照组相比,高剂量组能降低血清中MDA含量。与正常对照组相比,低、中剂量组能提高SOD的活力,而高剂量组与正常组无统计学差异。说明造模因素能增加IgE的表达,低、中剂量经验方药可降低其表达,而高剂量作用不明显。高剂量经验方药可降低MDA含量,而低、中剂量可提高SOD活力;12)从主证动物模型给药组外周血T淋巴细胞经时变化可见,与0d相比:主证大鼠模型给药组外周血CD3+细胞百分含量升高,具有统计学意义（P=0.002）;CD4+细胞百分含量随时间先下降后升高,6d时的差异具统计学意义（P=0.000）;CD8+细胞百分含量24d时升高,差异具有统计学意义（P=0.014）;CD4+/CD8+比值随时间变化先降后升、6d时差异具有统计学意义（P=0.004）、24d时差异具有统计学意义（P=0.013）;淋巴细胞总数随时间变化的差异不具有统计学意义。对比主证动物模型外周血T淋巴细胞经时变化可知,经验方药可调节造模因素引起的CD4+/CD8+比值下降;13)西北燥证兼心肾阴虚实验动物的外周血T淋巴细胞分类计数可见:与正常对照组相比,兼证大鼠模型组外周血CD3+细胞百分含量随时间先降后升,24d时的差异具有统计学意义（P=0.03）;CD4+细胞百分含量随时间先下降后升高,与正常对照组的差异不具统计学意义,但6d至24d的差异具统计学意义（P=0.042）;CD8+细胞百分含量的经时变化不具有统计学意义;CD4+/CD8+比值随时间变化下降、6d时差异具有统计学意义（P=0.004）。淋巴细胞总数随时间先升高后下降,其中0d和6d时的差异具有统计学意义（P<0.05）;14)析因设计分析阴虚造模因素与西北燥证造模因素,以fT3和fT4的变化进行考察,方差分析的结果表明,两因素及两因素的交互作用对大鼠血清中游离T3含量的影响无统计学意义（F=0.752,P=0.398）。而阴虚造模因素对游离T4含量的影响有统计学差异（F=5.592,P=0.030）,主证造模因素对游离T4含量的影响无统计学差异（F=3.618,P=0.074）,两因素的交互作用对大鼠血清中游离T4含量的影响有统计学意义（F=26.413,P=0.000）;15)与正常对照组相比,西北燥证兼支气管哮喘组与单纯支气管哮喘组使血清IgE水平升高,而给药组与正常组相比无统计学差异。与正常对照组相比,西北燥证兼支气管哮喘组与单纯支气管哮喘组血清IL-4水平升高,而模型给药组与正常组相比无统计学差异。血清IFN-γ在各组间未见明显差异。结论:1)西北燥证是方域性明显、以燥邪为主病因多元、病机错杂累及多脏的复合型中医证候;2)时空结合人工气候模拟实验设计能客观科学地反映西北燥证外感燥火风寒病因的实际情况,所制作的SD大鼠动物模型符合中医理论、切实可行;3)西北燥证造模因素施加最佳时间为4个气化周期（24d）。在此期间,模型动物饮水量明显增加、饮食量减少、体重增长缓慢;模型动物肺及颌下腺无明显病理学改变;4)进一步的微观指标观察可见,西北燥证主证造模因素可致:（1）外周血细胞分类计数正常,CD4+/CD8+比值随时间下降;（2）IL-1β水平降低,性激素E₂、T水平降低;（3）透射电镜下观察细胞内超微结构可见细胞膜损伤、嗜酸性粒细胞浸润及凋亡细胞;（4）TUNEL检测到模型动物肺及颌下腺凋亡表达,并以免疫组化方法检测到促凋亡蛋白bax、fas-l的高表达;5)对西北燥证模型给予经验方药的治疗,从实验结果可见:（1）西北燥证主证造模可致模型动物HPA轴紊乱,而低、中、高剂量的经验方药对其分别具有一定的调节作用;（2）低、中剂量经验方药可降低模型动物血清IgE水平,高剂量经验方药可降低血清中MDA含量,同时低、中剂量能提高SOD活力,表明经验方药具有抗过敏、抗氧化、清除自由基的能力,且其作用与药物浓度有关;（3）西北燥证主证造模可致模型动物CD4+/CD8+比值随时间下降,而给药（中剂量）后能使下降趋势明显;6)建立西北燥证兼心肾阴虚模型,初步考察主证造模因素与兼证造模因素之间存在交互作用;7)建立西北燥证兼支气管哮喘模型,经验方药可降低模型动物血清IgE、IL-4水平,认为经验方药治疗支气管哮喘的机理,部分是通过减少IgE、IL-4的合成而达到的。更多还原

【Abstract】 Objective: to design the northwest dryness syndrome animal model,the macroscopic syndrome differentiation index and experience formulas disproof evaluation model,at the same time,on the model animals with partial index for testing and analysis of modern medicine,the northwest dryness syndrome and its correlation disease mechanism of modern medicine and drug prevention study deeply to the microscopic level.Methods: 1)The system summary since northwest dryness syndrome concept is put forward to carry out the research work,from the medical concept of overall,analysis of the multiple causes of northwest dryness syndrome,combing the complexity of northwest dryness syndrome syndrome structure,so as to reveal its pathogenesis essence,establish the basic treatment of northwest dryness syndrome and treatment,the formulas for the northwest dryness syndrome syndrome model study to lay a theoretical basis.2)On the basis of northwest dryness syndrome etiology,symptoms and the biological characteristics of common laboratory animals,choose suitable for the northwest dryness syndrome animal model research with strain.3)Draw lessons from the six evils of the past literature of animal building experience,based on the northwest dryness syndrome causes the results of the study,using its dry fire wind chill with actual load factor relationship between climate index of reference from the northwest dryness syndrome prone areas of hotan actual statistics by climate dry fire wind chill constituent ratio,and selected laboratory artificial climate index and classification,composition,quantitative and combined with time and space transform regulation of artificial climate index factors,establish experimental scheme.4)Analyze the four corresponding relationship of the clinical and animal representation of northwest dryness syndrome from the body(metrology)syndrome differentiation and syndrome differentiation to make the transition to animal syndrome differentiation,to establish the northwest dryness syndrome animal measurement dialectical method.5)Experimental animals were randomly divided into normal group,model group and treatment group the formulas for eight gasification cycle(a cycle is 6d)the causes of simulated building.6)With the northwest dryness syndrome animal differentiation of measurement scale as the main evaluation index model,choose water,diet and stool properties,weight combined with disproving evaluation of animal models,the formulas and establishing building gasification cycles required.7)Animal models with optical microscope,lung,submandibular gland tissues,lung,animal models with transmission electron microscope,submandibular gland tissue cell structure,evaluation of representative organs in building element under the action of damage degree.8)Using flow cytometry test peripheral blood T lymphocytes in the change in the process of building,suggests that during the period of building the dynamic change of T lymphocyte subsets.9)TUNEL method in detecting lung experimental animal representative organs and the status of the submandibular gland tissue cell apoptosis and evaluation mode that the damage degree of factors on the experimental animals.10)Immune histochemical method in detecting lung experimental animals and submandibular gland apoptosis related proteins the BCL-2,the expression of Bax,fas-l,preliminary explore the possible pathway of apoptosis.11)ELISA method to detect peripheral blood and promote experimental animals adrenal hormone(ACTH)and Cortisol(Cortisol),catecholamine(CA)content,evaluation of building factors of neuroendocrine immune network HPA axis.12)Associates method is used to determine serum malondialdehyde(MDA)concentration and xanthine oxidase method determining superoxide dismutase(SOD)activity,evaluation of building elements.13)With flow cytometry test peripheral blood T lymphocytes in the building process to experience the change of the treatment,the formulas that experience effect on T lymphocyte subgroup animal models with the formulas of dynamic change.14)With "dry,fire,wind,cold," gasification ark(i.e.,the northwest dryness syndrome main building factors)joint lavage thyroxine method to establish the northwest dryness syndrome animal model of heart and kidney Yin deficiency,flow cytometry test peripheral blood T lymphocyte by change,and the factorial design makes a research on the forming die factors influence of fT3,fT4.15)"dry,fire,wind,cold," ark of gasification combined egg albumen sensitization legal system for northwest dryness syndrome and bronchial asthma model,and ELISA method to detect serum IgE experimental animals,IL-4,IFN-gamma level,evaluate the role of two factors and experience formulas.Results: 1)team in the past study revealed a northwest dryness syndrome is the external cause to dry,fire,wind,cold,internal cause in dry sensitive physique condition,and appear inside and outside the hair dry permit and multifarious syndrome;According to the principle of dialectical treatment selected northwest dryness syndrome main northwest dryness syndrome and heart and kidney Yin deficiency syndrome and the northwest dryness syndrome experience formulas of bronchial asthma.2)The selected temperature,humidity,light,wind,dust and other five factors for building artificial climate indicators,and used a modified artificial climate box,adding dust experiment box,at the same time as the five artificial climate index classification quantification assignment to the combination and dry fire wind cold properties,and formulate gasification cycle state formula,artificial climate index factors in time and space transform regulation plan,select SD rats for northwest dryness syndrome main animal model building.3)compared with control group,model group rats after building factors exert namely appear the phenomenon of water quantity to increase,and continues until the end of treatment.In dealing with the three gasification cycle before eating less obvious,however,as the processing of applying,to 8d,model group diet is no statistical difference compared with normal control group.Compared with control group,model group rats weight at the beginning of the applying process of 4~6d weight slow growth,P=0.001(P<0.05)difference is statistically significant,continue to 10~12d;After 13 d and model group compared with the control group weight differences were not statistically significant.Compared with the control group,model group rats during building factors exert always have constipation.4)The pathological tissue section HE dyeing,24 d and lung and submandibular gland tissue of previous models did not see obvious pathological changes,48 group d pathological damage of individual animals began to appear more apparent.5)Peripheral blood cell count,according to the results of each experimental animal blood count within the normal range,compared with normal control group,24 d,elevated monocytes ratio(Mon %),percentage of eosinophils(Eos)% lower statistically significant.6)From the main syndrome animal model of peripheral blood T lymphocyte by the changes can be seen,when compared with normal control group: CD3+,CD4+,CD8+cells percentage change over time,but no statistically significant difference,the CD4+/CD8+ratio decline over time,6d differences statistically significant(P=0.019),shows that building factors can reduce the CD4+/CD8+ratio.7)Experimental animal lungs observed under transmission electron microscope and submandibular gland tissue structures in the cell,the model group,a significant phenomenon of apoptosis,cell membrane damage,cell swelling,alveolar visible between eosinophil infiltration.8)TUNEL experiment result shows model group of alveolar,around the bronchi and submandibular gland serous cell nuclei have obvious yellow to brown staining of apoptotic cells.9)Immune histochemical method detecting experimental animal lungs and submandibular gland apoptosis related proteins expression: model group the BCL-2 expression is not obvious,bax,fas-l have more expression,that building factors may result in promoting apoptosis protein expression level.10)ELISA to detect peripheral blood serum ACTH,Cortisol,and CA content in experimental animals,compared with normal control group,the model of rat serum adrenocorticotropic hormone(ACTH)content in indifference,and Cortisol(Cortisol)content decreased(P=0.020),catecholamine(CA)content is also reduced(P=0.019).Compared with model group,low,medium and high doses of the drug group can improve the serum catecholamine(CA)content(P=0.028,P=0.028,P=0.000).At the same time low,medium and high doses of the drug group can improve the level of Cortisol(Cortisol)levels back to normal,and the model group differences statistically significant(P=0.007,P=0.007,P=0.000).Medium and high doses of the drug can increase the content of ACTH,compared with the model group with statistical significance(P=0.034,P=0.034).11)Compared with normal control group,model group rats serum IgE levels,give a low dose and dose after the formulas in the lower-and there was no difference in the normal control group,high dose drug group rats serum IgE not seen is reduced,to higher levels compared with normal control group,with statistical significance.Compared with normal control group,high dose group can reduce the MDA content in serum.Compared with normal control group,low,middle dose group can enhance the vitality of SOD,no statistical difference and high dose group and normal group.Building factors can increase the expression of IgE,low,medium dose experience can reduce the expression,the formulas and high dose effect is not obvious.Experience in high doses can reduce MDA content,the formulas and low,medium dose can improve SOD vigor.12)From the main syndrome animal model for groups of peripheral blood T lymphocyte by the changes can be seen,when compared with 0d: the rat model with master card to medicine group peripheral blood CD3+cells percentage increases,statistically significant(P=0.002).CD4+cells percentage increases with time after the first drop,the difference of 6d statistical significance(P=0.000);Higher percentage of CD8+cells at 24 d,difference have statistical significance(P=0.014);CD4+/CD8+ratio change with time after the first drop rose,6d differences statistically significant(P=0.004),24 d statistically significant difference(P=0.013);The total number of lymphocytes no statistically significant difference in change over time.Compared to peripheral blood T lymphocyte by main syndrome animal model of change,the empirical formulas made mold adjustable factors of CD4+/CD8+ratio decreased.13)The northwest dryness syndrome of heart and kidney Yin deficiency of experimental animals peripheral blood T lymphocyte count: compared with normal control group,and the model group rats peripheral CD3+cells percentage to fall after rise over time,24 d difference have statistical significance(P=0.03);CD4+cells percentage increases with time after the first drop,no statistical significance difference with the normal control group,but 6d to 24 d difference statistical significance(P=0.042);Percentage of CD8+cells when the change is not statistically significant;CD4+/CD8+ratio drop change over time,6d differences statistically significant(P=0.004).Total number of lymphocytes fall after rise over time,including 0d and 6d differences statistically significant(P<0.05).14)The factorial design analysis of Yin deficiency made die factors with northwest dryness syndrome factors,with the change of FT3 and FT4,the results of variance analysis showed that the interaction of the two factors and two factors of the effect of free T3 content in the serum of rats with no statistical significance(F=0.752,P=0.752).And Yin deficiency building factors influence on free T4 content was statistically difference(F=5.592,P=5.592),the main card building factors influence on free T4 content no statistical differences(F=3.618,P=3.618),the interaction of the two factors on the impact of free T4 content in serum of rats was statistically significant(F=26.413,P=26.413).15)Compared with normal control group,the northwest dryness syndrome and bronchial asthma with pure bronchial asthma group make serum IgE levels,while no statistical differences compared to medicine group and normal group.Compared with normal control group,the northwest dryness syndrome and bronchial asthma with pure bronchial asthma group had elevated levels of serum IL-4,to medicine group and model no statistical difference compared with normal group.Serum levels of IFN-gamma no significant differences between groups.Conclusion: 1)Northwest dryness syndrome is square performance significantly,mainly dry evil cause,pathogenesis of involvement,how dirty mixed complex syndromes.2)The time and space in combination with artificial climate simulation experimental design can objectively reflect the northwest dryness syndrome scientifically exogenous dryness of the actual situation of fire wind cold causes made SD rat animal model conforms to the theory of traditional Chinese medicine,is feasible.3)The northwest dryness syndrome building factors exert the best time for four gasification cycle(d)24.Water quantity increased significantly during this period,the animal model,eating less,slow weight gain;Model animal lungs and submandibular gland has no obvious pathological changes.4)Further the microscopic index observation,northwest dryness syndrome main building factors can cause:(1)the classification of peripheral blood cell count is normal,the CD4+/CD8+ratio decline over time.(2)the cell membrane were observed under tem cell ultrastructure of visible damage,eosinophil infiltration and cell apoptosis.(3)animal models with TUNEL detect lung and submandibular gland apoptosis expression,and to promote apoptosis proteins bax immunohistochemical method to detect and high expression of fas-l.5)The northwest dryness syndrome model experience of the formulas was given from the experiment result shows:(1)the northwest dryness syndrome main building HPA axis is disorder,can cause animal model and the experience of high,medium and low dose the respectively the formulas have certain adjustment.(2)low,medium dose experience can reduce serum IgE level model animals,the formulas of high dose experience can reduce the MDA content in serum,the formulas at the same time,low dose can improve SOD vigor,showed that experience formulas have allergy,antioxidant,the ability of scavenging free radicals,and its role is associated with drug concentration.(3)the northwest dryness syndrome main building can cause animal model of CD4+/CD8+ratio decline over time,and after dosing(dose)can make the downward trend is obvious.6)Build the northwest dryness syndrome,heart and kidney Yin deficiency model preliminary study the main syndrome and building factors and interactions between building factors.7)Establishment of northwest dryness syndrome and bronchial asthma model,experience can reduce serum IgE animal models with the formulas,IL-4 levels,experience that the mechanism of treating bronchial asthma with the formulas,partly by reducing the synthesis of IgE,IL-4 and reach.更多还原