【作者】 卢志刚；

【导师】 王亚利；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2010， 博士

【摘要】 季节气候是人类赖以生存的基本条件,其变化也容易导致疾病的发生,尤其对呼吸系统疾病的发生影响比较显著。季节气候的变化刺激机体后,免疫系统必将起到相应的免疫防御和免疫自稳作用,季节变化影响到呼吸系统时会引起局部免疫平衡的波动,如果超过人体生理范围将导致呼吸系统疾病的发生。呼吸系统免疫调节是一个精细的受严格调控的过程。存在于呼吸系统的固有和适应性免疫机制共同抵御不利因素的侵袭,保证了呼吸系统正常生理功能的进行。研究发现,呼吸系统疾病的季节易感性与人类抵抗感染性疾病的基础—-免疫功能的季节性调节紊乱关系密切,如果免疫系统这种的季节性变化规律及其机制能被发现,将会在呼吸系统疾病的防治方面产生重大的影响。因此,了解呼吸道免疫自稳状态及其调控机制的季节性变化规律对提高呼吸系统疾病的诊断、治疗及预后判断具有重要意义。在呼吸系统免疫防御功能中,行使固有免疫防御功能的免疫相关物质,如自然杀伤淋巴细胞、干扰素、细胞因子和行使适应性免疫防御功能的免疫相关物质,如T淋巴细胞以及与呼吸系统防御相关的免疫分子,如肺表面活性物质都成为人们的研究热点。“天人相应”理论是中医学理论体系的重要组成部分,该理论认为人和自然环境是一个有机整体,两者的运动变化可直接或间接地相互影响,机体也相应地随自然变化出现生理或病理上的反映。随着现代医学神经神经-内分泌-免疫网络的发现,人们认识中医肺脏整体功能的与西医学呼吸系统之间存在者很多相似之处,中医对卫气的认识也与现代医学的免疫功能类似等。中医学认为肺卫功能正常是机体防御病邪的重要基础。这为从中西医结合角度研究它们之间的关系提供了很好的桥梁和纽带。目前从临床实践中可证实“天人相应”理论的科学性,尤其是肺与季节气候发病的关联性,但是季节如何影响人体呼吸系统免疫功能的,其免疫物质基础尚不十分清楚。近年来,许多学者对“天人相应”理论的科学内涵从各个角度进行了探讨,尤其是随着现代免疫学的发展,人们从免疫学的角度发现中医的这些理论与现代医学的一些研究结果不谋而合,季节变化对呼吸系统免疫功能影响以及调节作用机制方面的研究虽然取得一些进展,但具体机制仍不明确。本课题以“天人相应”理论和肺卫理论为基础,就季节变化对呼吸系统免疫物质的影响提出假设,以期验证其与免疫物质的活动变化相关性,为临床上常见的呼吸系统疾病季节性发病提供一定的实验依据,同时在预防和保健领域亦具有重要的参考价值。本实验内容主要包括以下三部分:第一部分:季节变化对健康大鼠肺组织IL-1?、IL-2、IL-6、IL-10和SP-A表达的影响目的:通过观察季节变化过程中大鼠肺组织IL-1?、IL-2、IL-6、IL-10和SP-A的表达,探讨季节变化对大鼠肺脏免疫调节的作用机制,为呼吸系统季节性发病的病理生理机制的认识提供实验依据。方法:清洁级健康3月龄雄性SD大鼠64只,体质量180-220克,分别按购买时间分为立春、春分、立夏、夏至、立秋、秋分、立冬、冬至8个组(n=8)。分别于节气前14天购入实验大鼠。自由摄取水及普通鼠全价颗粒饲料,室温饲养,接受自然光照。饲养到相应节气,于当日下午6时将动物断头处死,取出大鼠全肺组织进行IL-1?、2、6、10和SP-A mRNA检测。结果:2季节变化对健康大鼠肺组织IL-1?表达的影响在正常生理状态下,全年健康大鼠肺组织中IL-1? mRNA表达呈现出夏季较高,春秋冬季较低(立夏>春分>夏至>立春>立冬>冬至>立秋>秋分)的变化趋势。半定量PCR结果显示春分、立夏、夏至相对表达水平,较其他组均有显著性升高(P<0.05或P<0.01)。秋冬季节各组间相互比较无显著性差异(P>0.05)。3季节变化对健康大鼠肺组织IL-2表达的影响在正常生理状态下,全年健康大鼠肺组织中IL-2 mRNA表达呈现出春夏季较高,秋冬季较低(春分>夏至>立夏>立春>冬至>立秋>秋分>立冬)的变化趋势。半定量PCR结果显示与春分、立夏组和夏至组相对表达水平比较,其他组明显降低(P<0.01)。秋冬季节各组间相互比较无显著性差异(P> 0.05)。4季节变化对健康大鼠肺组织IL-6表达的影响在正常生理状态下,全年健康大鼠肺组织中IL-6 mRNA表达呈现出夏季较高,冬季较低,春秋次之(夏至>立夏>立秋>立春>春分>秋分>立冬>冬至)的变化趋势。半定量PCR结果显示与立冬和冬至组比较,立春、春分、立夏、夏至和立秋组显著性升高(P<0.05或P<0.01);与夏至组比较,春分、秋分、立冬和冬至组显著性降低(P<0.05或P<0.01);与立秋组比较,秋分、立冬和冬至组显著性升高(P<0.05)。5季节变化对健康大鼠肺组织IL-10表达的影响在正常生理状态下,全年健康大鼠肺组织中IL-10 mRNA表达呈现出夏秋季较高,冬春较低(夏至>秋分>立秋>立冬>立夏>春分>立春>冬至)的变化趋势。半定量PCR结果显示与夏至组比较,立春组和冬至组显著性降低(P<0.05)。6季节变化对健康大鼠肺组织SP-A表达的影响在正常生理状态下,健康大鼠肺组织中SP-A mRNA在春夏季节明显增加,秋冬季节中SP-A mRNA表达较少(夏至>春分>立夏>立春>立秋>秋分>冬至>立冬)的变化趋势。半定量PCR结果显示与春分和夏至组比较,立秋、秋分、立冬和冬至组显著性降低(P<0.05或P<0.01);与立冬组比较,其他各组均显著性升高(P<0.05或P<0.01)。结论:季节通过调节大鼠肺组织中IL-1?、2、6、10和SP-A的含量的变化来影响肺的免疫防御功能,其中IL-1?、2、6、10和SP-A可能是机体免疫功能季节性变化的中介物质基础。肺部免疫防御功能大致存在春夏较高,秋冬季低的季节节律,这可能是秋冬季节呼吸系统多发病的病理生理学基础之一。第二部分:季节变化对健康大鼠血清Th1和Th2型细胞因子的调节作用及肺组织形态学的影响目的:本部分研究通过建立自然环境下大鼠动物模型,旨在通过观察不同季节气候条件下大鼠血清中Th1和Th2型细胞因子(IL-10和INF-γ)含量的变化以及肺组织形态学的改变,以期阐释季节变化影响呼吸系统中Th1和Th2免疫平衡的调节作用及其机制,为指导临床实践提供一定的理论依据。方法:动物分组及标本取得同第一部分。组织学观察:动物断头后,迅速开胸取右肺中叶,用4%多聚甲醛固定,脱水、包埋、切片,苏木精-伊红HE染色,镜下观察形态学改变。IL-10和IFN-γ水平的检测:大鼠血清离心后,取上清,采用双抗体夹心ABC-ELISA法测定血清中的IFN-γ水平,放射免疫分析法检测IL-10水平。结果:1季节变化对健康大鼠肺组织形态学的影响春夏秋季组:大鼠肺组织支气管内及周围结构连续,无明显炎性细胞浸润,组织黏膜未见充血水肿渗出及肺泡间隔狭窄,肺泡腔清亮宽敞无扩大。立冬组和冬至组:可见肺泡腔变窄,肺泡间隔变大,无明显炎性细胞浸润以及黏膜充血水肿。2季节变化对正常大鼠血清Th1型细胞因子IFN-γ含量的影响在正常生理状态下,全年健康SD大鼠血清中IFN-γ呈现出冬季较低,其他季节较高(立夏>春分>立春>立秋>秋分>夏至>冬至>立冬)的变化趋势。与立冬组和冬至组比较,其他各组均显著性升高(P<0.05或P<0.01);立夏组较夏至组、立秋组、秋分组、立冬组和冬至组均显著性升高(P<0.05或P<0.01);春分组较夏至组、秋分组、立冬组和冬至组均显著性升高(P<0.05或P<0.01)。3季节变化对正常大鼠血清Th2型细胞因子IL-10含量的影响在正常生理状态下,全年健康SD大鼠血清中IL-10呈现出夏季较高,其他季节较低(立夏>夏至>立冬>立春>冬至>立秋>春分>秋分)的变化趋势。与秋分组比较,其他各组显著性升高(P<0.05或P<0.01)。春夏冬季节间比较无显著性差异(P>0.05)。结论:季节变化过程中大鼠血清中IFN-γ和IL-10的含量发生了季节性变化,季节通过调节它们的含量来影响机体Th1/Th2免疫平衡。冬季机体可能通过降低IFN-γ含量,升高IL-10含量,降低呼吸系统免疫功能,导致多种呼吸系统疾病疾病的发生。第三部分:季节变化对健康人外周血CD3+、CD4+、CD8+和CD56+淋巴细胞比率的影响目的:通过在季节不同时点对健康人外周血CD3+、CD4+、CD8+和CD56+淋巴细胞的检测,探讨季节变化对人体免疫功能的影响,为“天人相应”理论应用于临床呼吸系统疾病的防治提供确切的客观指标。方法:选取我校2007级新入学的学生志愿者20名,男女各10例,均经我校附属医院体检合格,无各种感染、无急慢性口腔鼻咽疾病、肿瘤、免疫系统疾病或肝肾功能不全等病史,且在实验期间内未使用抗生素、非甾体类消炎药及影响免疫系统功能的药物。分别于8个节气不同实验日前7天充分休息且处于室内外自然环境状态下,于实验日下午在安静空腹状态下抽取2mL外周血。应用流式细胞术对外周血中CD3+、CD4+、CD8+、CD56+淋巴细胞进行分析。结果:1季节变化对健康人外周血CD3+淋巴细胞数量的影响全年健康人外周血中CD3+T淋巴细胞比率呈现出夏季较高,冬季较低,春秋季节次之(夏至>立夏>立秋>立春>春分>秋分>立冬>冬至)的变化趋势。与立夏组和夏至组比较,立冬组和冬至组CD3+T淋巴细胞比率均显著性下降(P<0.01)。2季节变化对健康人外周血CD56+淋巴细胞数量的影响全年健康人外周血中CD56+自然杀伤淋巴细胞比率呈现出夏季较高,春秋冬季节较低(立夏>夏至>春分>立冬>立秋>立春>秋分>冬至)的变化趋势。各季节组未见显著性差异(P>0.05)。3季节变化对健康人外周血CD4+T淋巴细胞比率的影响全年健康人外周血中CD4+T淋巴细胞比率呈现出夏季较高,冬季较低,春秋季节次之(夏至>立夏>春分>立春>立秋>秋分>冬至>立冬)的变化趋势。与立夏组和夏至组比较,立冬组和冬至组CD4+T细胞均显著性下降(P< 0.05)。4季节变化对健康人外周血CD8+T淋巴细胞比率的影响全年健康人外周血中CD8+T淋巴细胞比率呈现出冬季较高,夏季较低,春秋季节次之(冬至>立秋>立春>立冬>秋分>春分>立夏>夏至)的变化趋势。与夏至组比较,冬至组CB8+T细胞比率均显著性上升(P<0.05)。5季节变化对健康人外周血CD4+/ CD8+ T细胞比值的影响全年健康人外周血中CD4+/ CD8+T细胞比值呈现出夏季较高,冬季较低,春秋季节次之(夏至>立夏>春分>立春>立秋>秋分>立冬>冬至)的变化趋势。与立夏组比较,冬至组CD4+/ CD8+比值显著性下降(P<0.05);与夏至组比较,立秋组、秋分组、立冬组和冬至组CD4+/ CD8+比值显著性下降(P<0.05或P<0.01)。结论:季节变化对机体细胞免疫功能具有一定的调节作用,免疫功能存在春夏季强秋冬季弱的变化趋势,与中医天人相应和肺卫防御理论的认识是一致的。春夏季可能通过调节人体外周血中CD3+、CD4+、CD8+T细胞数量,使CD4+T细胞升高,而CD8+T细胞降低,上调CD4+/CD8+比例,起到免疫增强作用。更多还原

【Abstract】 Seasonal climate is the basic conditions of human life,its changes are most likely to lead to diseases,especially respiratory diseases(RD).After body were stimulated by seasonal variation(SV),the immune system will play the role of immune defense and immune homeostasis.The partly immune balance of the affected lungs will fluctuated by SV,which will lead to RD if beyond physiological range.The immunity poikilostasis of respiratory system(RS) was a delicate process strictly regulated.The RS mechanism of inherent and adaptive immunity ensured the conduct of normal physiological function of RS by against the common attacks of adverse factors.Once this seasonal physiological and pathological transform rule were discove,the great contribution will be appeared in prevention and cure of RS. Therefore, understanding the respiratory immune homeostasis and its regulatory mechanisms by SV would had great significance if improving the level of diagnosis,treatment and prognosis on respiratory systemic diseases involved pulmonary infection.In the RS function of the immune defense,the immune related substances of inherent immune defense,such as natural killer lymphocytes,interferon,and cytokine;adaptive immune defense,such as T lymphocytes and other immunity molecule related RS defense,such as pulmonary surfactant(PS) had became a research focus.The theory of human-environment Inter Relation(HEIR) is one of the most important content of TCM,which consider that the inner lives of people recognize activities and the natural environment,social environment exists between interrelated,mutual influening complex relationship,the body occurs naturally physiological or pathologica reflect with a corresponding change.As soon as nerve-endocrine-immune regulation theory been discovered,pepoles realized that they have many similaties,particularly in the medical understanding of the lung organs function and West respiratory system,and the Wei-qi function and immunity defense function so it provide us a good link and bridges to research.At present the scientific HEIR theory had been confirmed by clinical practice,especially in lung and seasonal climate pathogenesis,but how SV affects the body’s RS immune function and its material basis is not yet clear.In recent years,the scientific connotation of HEIR theory was discussed from various angles,particularly with the development of modern immunology, people found the resemblance between these TCM theory and modern medical investigation from immunology.A number of advancement had been made in SV on immune function and mechanism of the lungs,but the specific mechanism remains unclear.Based on the theory of HEIR and Fei-- Wei,this subject pose the hypothesis of SV on the RS immune substances,verify the changes and immune substances closely related activities,provided experiment base for the clinically common high seasonality prevalence of RS,meanwhile for preclinical and physical fitness medicine.The content of this study were divided into three parts.Part I:Effect of seasonal variation on expression of interleukin-1?,2,6,10 and surfactant associated protein A in healthy rat lung tissuesObjective:By observing the expression changes of IL-1?,2,6,10and SP-A of male rats in each season,to explore the effects of seasonal changes on the immunity of lung of normal rats,and to provide new ideas for the experimental basis to the cognition of pathology and physiology mechanism for seasonal attack of RS diseases.Methods: Sixty-four SD male rats, weighted 180-220g, purchased from forteen days before each solar term and divided into groups in chronological order,were randomly divided into eight groups(n=8):Vernal Begins(VB) groups,Vernal Equinox(VE) group,Summer Begins(SB) group,Summer Solstice(SS) group,Autumn Begins(AB) group,Autumn Equinox(AE) group,Winter Begins(WB) group and Winter Solstice(WS) group.All rats were housed with normal forage and drank freely under room temperature,receiving natural light. Then the rats were sacrificed by decapitation before 18 o’clock at corresponding solar term,then the IL-1?,2,6,10 and SP-A mRNA expression changes of the lung tissue in rats from each group were detected by RT-PCR method.Results 1 Effect of seasonal variation on expression of interleukin-1? in healthy rat lung tissuesThe immune materials of lung in different group were higher in spring and lower in other seasons.The groups that SB>VE>SS>VB>WB>WS>AB>AE changes in the trend.The expression of IL-1? in VE,SB and SS group was significantly higher than others groups(P<0.05 or P<0.01).There was no significant difference betwee the all spring and winter groups(P>0.05).2 Effect of seasonal variation on expression of interleukin-2 in healthy rat lung tissuesThe immune materials of lung in different group were higher in spring and lower in other seasons.The groups that VE>SS>SB>VB>WS>AB>AE>WB changes in the trend.The expression of IL-1? in VE,SB and SS group was significantly higher than others groups(P<0.01).There was no significant difference between the all spring and winter groups(P>0.05).3 Effect of seasonal variation on expression of interleukin-6 in healthy rat lung tissuesThe immune materials of lung in different group were higher in summer and lower in winter.The groups that SS>SB>VB>AB>AE>VE>WB>WS changes in the trend. Compared with WB and WS group,the expression of IL-6 was significantly higher in VB,VE,SB,SS and AB group(P<0.05 or P< 0.01);Compared with SS group,it was significantly lower in VE,AE,WB and WS group(P<0.05 or P<0.01);Compared with AB group,it was significantly lower in AE,WB and WS group(P<0.05 or P<0.01).4 Effect of seasonal variation on expression of interleukin-10 in healthy rat lung tissues The immune materials of lung in different group were higher in summer /autumu and lower in spring/winter.The groups that SB>VE>SS> VB>WB >WS>AB>AE changes in the trend. Compared with SS group,the expression of IL-10 was significantly lower in VB and WS groups(P<0.01). 5 Effect of seasonal variation on expression of SP-A in healthy rat lung tissues The immune materials of lung in different group were higher in spring /summer and lower in autumn/winter.The groups that SS>VE >SB>VB >AB >VE>WS>WB changes in the trend.Compared with VE and SS group,the expression of IL-10 was significantly lower in AB,AE,WB and WS groups (P<0.05 or P<0.01);compared with WB group,it was significantly higher in other groups(P<0.05 or P<0.01).Conclusion:The expression of IL-1 ?,2,6,10 and SP-A were adjusted by SV, which effected the immune function of lung.The immune materials of IL-1 ?,2,6,10 and SP-A maybe the mesomerism material foundation of the the immune function.The mmune function of lung existed the seasonal rhythm of lower in autumn and winterr and higher in spring and summer. It may be one of the pathophysiologic basis of respiratory diseases mostly happened in autumn and winter.Part II:Effects of SV on regulatory role of Th1 and Th2 cytokines and lung morphological changes of the SD ratsObjective:On the basis of establishing the rat model in the natural environment, we designed to observe the climatic conditions in different seasons of IL-10,INF-γin SD rat serum and lung histomorphology,expected to explain influencing the Th1/Th2 immune balance in the RS and its mechanism for guiding clinical practice and providing a theoretical basis. Methods:Animal grouping,making model and the statistical methods of data are same as part I.Morphological observation:after sacrificed by decapitation,the middle lobe of the right lung was taken and fixed with 4% paraformaldehyde and gave the dehydration,embedding and sections,then,the hematoxylin-eosin HE staining.Testing methods of INF-γand IL-10:the prepared serum was given the centrifugation,after that,the supernatants was extracted and gave subpackage.IFN-γlevel were measured with the double -antibody sandwich ABC-ELISA method,and IL-10 Radioimmunoassay.Results1 Effects of SV on lung morphological of the SD ratsSping/summer/autumn groups:there was no obvious inflammatory cell infiltration from the bronchus of lung tissues and surroundings in rats with intact wall,there were no congestion and edema in the tissue mucosa,and the alveolarcavity was clear and spacious. Winter groups:The alveolar lumen narrowed with the widening of alveolar septum,there were no the congestion and edema of tissue.2 Effects of SV on the content of Th1 cytokines of the SD ratsThe immune materials IFN-γof rat serum were lower in winter and higher in other groups.The groups that SB>VE>VB>AB>AE>SS>WB>WS changes in the trend.Compared with WB and WS groups, it was significantly higher in the other groups(P<0.05 or P<0.01);Compared with SB group,it was significantly lower in SS,AB,AE,WB and WS groups(P<0.05 or P<0.01); Compared with VE group,it was significantly lower in SS,VE,WB ana WS groups(P<0.05 or P<0.01).3 Effects of SV on the content of Th2 cytokines of the SD ratsThe immune materials IL-10 of rat serum were higher in summer and lower in other groups.The groups that SB>SS>WB>VB>WS>AB>VE>AE changes in the trend.Compared with AE group,the level of IL-10 was significantly higher in other groups(P< 0.05 or P<0.01). There was no significant difference between Spring and summer and winter groups(P>0.05). Conclusion:The content of IL-10 and IFN-γin rat’s blood serum occurred seasonal change,which were adjusted by SV to effect the Th1/Th2 immunologic balance.Winter could depress the content of IFN-γand heighten IL-10 to degrade the immune function of RS,which resulted in anumber RD.Part III:Influence of SV on the ratio of CD3+,CD4+,CD8+,CD56+ in Healthy IndividualsObjective:To observed one-year CD3+,CD4+,CD8+ and CD56+ lymphocyte in healthy individuals,and studied the effect of SV on immune function at the cellular level,in order to provide accurate and objective indicators for the HEIR theory applied to clinical prevention and treatment of RD.Methods:Twenty volunteers of Our School new-enrolled students in 2007 were chosed,men and women each 10 cases,which were confirmed by our school affiliated hospital.Those was healthy,free of all infection,acute and chronic oral nasopharyngeal disease,cancer,immune disorders,or liver and kidney dysfunction,and were not used antibiotics,non-steroidal anti- inflammatory drugs and drugs affecting the immune system in the experimental period.All volunteers had full rest in a state of indoor and outdoor environment before eight experiments time,and 2mL peripheral blood collected in a quiet afternoon under fasting conditions.Lymphocytesubsets by means of flow cytometry CD3+,CD4+,CD8+ and CD56+ lymphocyte in healthy individuals was determined.Results1 Effect of SV on CD3+in Healthy IndividualsThe immune materials CD3+ of healthy human serum were higher in summer and lower in winter.The groups that changes SS>SB>AB>VB>VE >AE>WB>WS >in the trend.Compared with SB and SS group,the expression of CD3+ was significantly lower in WB and WS groups(P<0.05 or P<0.01).2 Effect of SV on CD56+in Healthy IndividualsThe immune materials CD56+ of healthy human serum were higher in summer and lower in other groups.The groups that SB>SS>VE>WB>AB>VB >AE>WS changes in the trend.There was no significant difference betwee all groups(P>0.05).3 Effect of SV on CD4+in Healthy IndividualsThe immune materials CD4+ of healthy human serum were higher in summer and lower in winter.The groups that SS>SB>VE>VB>AB>AE>WS >WB changes in the trend.Compared with SB and SS group,the expression of CD4+ was significantly lower in WB and WS groups(P<0.01);There was no significant difference betwee spring and autumu groups(P>0.05). 4 Effect of SV on CD8+in Healthy IndividualsThe immune materials CD8+ of healthy human serum were higher winter in and lower in summer.The groups that WS>AB>VB>WB>AE>VE>SB>SS changes in the trend.Compared with SS group,the expression of CD8+ was significantly higher in WS groups(P<0.05).5 Effect of SV on the ratio of CD4+/ CD8+ in Healthy IndividualsThe ratio of CD4+/ CD8+ were higher in summer and lower in winter.The groups that SS>SB>VE>VB>AB>AE>WB>WS changes in the trend. Compared with SB group,the expression of CD4+/ CD8+ was significantly lower in WS groups(P<0.05);Compared with SS group,it was significantly lower in AB,AE,WB and WS groups(P<0.05 or P<0.01). Conclusion:There were a certain accommodation role by SV on immune functions,which tendency were strong in spring/summer weak in autumn/winter,with the corresponding recognition between Chinese HEIR theory and lung health awareness defense theory.Spring and summer could adjust the number of CD3+,CD4+,CD8+ T cell in blood serum,by heightening CD4+ and degrading CD8+,thus up-regulated the ratio of CD4+/CD8+ to emerge immunological enhancement.更多还原