不同外邪因素对小鼠免疫功能影响的研究
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摘要
目的:从《黄帝内经》(以下简称《内经》)生态医学思想理论出发,研究六淫致病性质、特点及发病规律。通过实验探讨不同外邪因素对正常及免疫低下小鼠一般情况和免疫状态的影响,寻找外邪致病与环境因素及机体状态之间的相关性,为现代生态病因学的研究奠定理论基础与实验依据,进一步为外邪致病的诊治提供新思路、新方法。
方法:
1分组:昆明种小鼠64只,雌雄各半,体重(20±2)g,随机分为8组,分别为:正常组、风寒组、寒湿组、湿热组、低免组、风寒低免组、寒湿低免组、湿热低免组,每组8只。
2造模:
(1)利用人工气候箱建立风寒、寒湿、湿热三种外邪环境模型,将小鼠置于相应外邪环境模型中,每天持续刺激8h,连续7天。每天观察记录小鼠精神状态、活动情况、毛色、皮肤关节及二便等情况,并定时记录小鼠进食及饮水量、体重。
(2)制备免疫低下模型:小鼠正常饲养2天后,各低免模型组给予正常小鼠腹腔注射环磷酰胺(CY)80mg/kg,连续3天。其余做法同上。
3检测:
(1)置于外邪环境中的每一天,观察小鼠一般情况的变化,并记录。
(2)环磷酰胺造模后,置于外邪环境连续7天后,取材。具体有小鼠摘眼球取血、离心,取血清检测小鼠免疫指标(血清中IL-2、IFN-γ含量变化);无菌取脾,MTT法检测脾淋巴细胞增殖能力。
结果:
1与正常组小鼠相比,各模型组小鼠一般情况发生改变,表现出一系列的病理症状。在整个造模期间各模型小鼠活动情况明显下降,出现精神萎靡,体重增长速度减慢或体重减轻,进食饮水量减少,出现稀便,毛色失去光泽,部分小鼠出现足背水肿及关节肿大,皮肤发红等。各低免模型组相比外邪模型组,其病理症状出现的时间更早,且较为严重。其中对进食饮水量的影响以寒湿、湿热外邪较为显著,对体重的影响以寒湿外邪较为显著。
2各模型组血清中IL-2、IFN-γ的含量及脾淋巴细胞增殖能力明显低于正常组,经统计学处理,均有显著性差异(P<0.05或P<0.01)。其中各低免模型组的下降程度更大。其中以寒湿外邪对血清中IL-2、IFN-γ的含量及脾淋巴细胞增殖能力的影响相对更为显著。
结论:
1从生态医学角度提出人体既要“天人合一”又要“正气存内”,方可“邪不可干”,指出疾病的发生是机体与外界环境相关性失衡所致,是多种因素致病。
2实验显示在长期“太过”气候的影响下,会出现机体的病理状态;不同外邪因素均会引起机体免疫功能的下降,即外邪导致正气不足使机体与外界环境状态失衡而发病;外邪对免疫低下小鼠其影响更为显著;综合实验结果,在长时间风寒邪气、寒湿邪气、湿热邪气的刺激中,以寒湿邪气刺激对机体免疫功能的影响更为显著。
Objective:
This thesis,beginning with ideological theory of Ecological Medicine of Huangdi Neijing (hereinafter referred to as Neijing), discovers the traits of disease and pathogenesis caused by six pathogenic factors. Furthermore, the thesis analyzes the effect of the general conditions and immune state on normal mice and immunodeficiency mice treated by different exogenous pathogens according to experiments, looking for relativity among diseases caused by exogenous pathogen, environmental factors and the state of organism. This research prepares the academic ground and experimental reference for the research of the modern ecology etiology, which provides new thoughts and methods for diagnosis and treatment of diseases caused by exogenous pathogen.
Methods:
1 Group dispensation: 64 Kunming mice, the amount of male and female mice being equal in, are divided into 8 groups as normal group, wind-cold group, damp-cold group, damp-heat group, immunodeficiency group, wind-cold immunodeficiency group, cold-damp immunodeficiency group and damp-heat immunodeficiency group in random. Each group contains 8 mice, weighting between (20±2) g.
2 Model:
(1) The mice were put into relevant exogenous pathogenic model of wind-cold, damp-cold and damp-heat environment and treated eight hours per day for seven days. Through the observation of the mice, the author recorded the conditions of energy, activity, coat color, skin, joint and urine and stool. At the same time, taking food and water consumption as well as the weight were recorded regularly.
(2) Preparation immunodeficiency model: The normal mice were normally bred 2 days, and then the mice were given injection of cyclophosphamide(CY)80mg/kg through abdomens in each immunodeficiency model,injecting CY continuously 3 days. The rest operating procedures are same as mentioned above.
3 Detection:
(1)The mice were put into the environment of exogenous pathogen each day, observed and recorded the change of general conditions.
(2)After establishing the model of cyclophosphamide, the mice, which in the environment of exogenous seven days, were extracted samples. The specific measures include operating procedures as follows. The blood of mice was taken by removing the eyeballs. The immune indicators of mice were tested by taking serum (the change of contents of IL-2、IFN-γin serum). The spleens of mice were taken aseptically, and then the ability of lymphocyte proliferation in spleen was tested with the method of MTT.
Trial results:
1 The general conditions of the mice of wind-cold, damp-cold and damp-heat groups have changed, which shows a series of pathological symptoms when compared with normal group. During the whole model, the conditions both mental and physical of mice in each group declined that mice appeared depressed, the increase of weight has been moderate, taking food and water consumption has been reduced, looser stools, and lusterless coat color. Furthermore the part of mice developed symptoms with swollen joint, redness of the skin and even ulcerated through the skin and so forth. The pathologic symptoms appear in the immunodeficiency group, which are earlier and seriously than other groups.
2 The level of IL-2 and IFN-γin serum and the ability of lymphocyte proliferation in spleen of the mice in each model are significantly lower than that of the mice in normal group, and there is significant discrepancy in statistics (P <0.05 or P <0.01). The test indexes of immunodeficiency group were much lower than that of other groups. The results showed that all the models of exogenous pathogen as wind-cold, damp-cold and damp-heat could cause decline of the immune function of organism and diseases.
The level of IL-2 and IFN-γin serum by experiment shows that there is significant change among wind-cold group, damp-cold group and damp-heat group of immunodeficiency group, in which the test indexes of wind-cold group and damp-cold group are lower obviously with comparison among groups. The ability of lymphocyte proliferation in spleen by experiment shows that there is significant discrepancy of wind-cold group and damp-cold group among immunodeficiency group.
Conclusion:
1 Based on the theoretical research of pathogenesis of six pathogenic factors, this thesis concludes that the occurrence of diseases is mainly due to such two factors as health qi in human body and exogenous pathogen (or six pathogenic factors). The mice are growing in natural climate under normal circumstances, while the mice appear pathological symptoms of organism under influence of beyond normal range of climate. Meanwhile, with points of view of Ecological Medicine, standpoints are presented that human body requires“Correspondence between Man and Nature”and“sufficient health qi inside the body”that“the pathogenic qi have no way to invade the health body”, and the cause of diseases are produced by several factors that is led by ecological pathogen.
2 Therefore the experiment has proved that different exogenous pathogen all can cause the decline of immunity of organism that means exogenous pathogen cause weakness of body to produce disease. During the long-playing stimulate amid wind-cold pathogenic factor, damp-cold pathogenic factor and damp-heat pathogenic factor, the influence for immunity of organism by damp- cold stimulate is more distinct.
方法:
1分组:昆明种小鼠64只,雌雄各半,体重(20±2)g,随机分为8组,分别为:正常组、风寒组、寒湿组、湿热组、低免组、风寒低免组、寒湿低免组、湿热低免组,每组8只。
2造模:
(1)利用人工气候箱建立风寒、寒湿、湿热三种外邪环境模型,将小鼠置于相应外邪环境模型中,每天持续刺激8h,连续7天。每天观察记录小鼠精神状态、活动情况、毛色、皮肤关节及二便等情况,并定时记录小鼠进食及饮水量、体重。
(2)制备免疫低下模型:小鼠正常饲养2天后,各低免模型组给予正常小鼠腹腔注射环磷酰胺(CY)80mg/kg,连续3天。其余做法同上。
3检测:
(1)置于外邪环境中的每一天,观察小鼠一般情况的变化,并记录。
(2)环磷酰胺造模后,置于外邪环境连续7天后,取材。具体有小鼠摘眼球取血、离心,取血清检测小鼠免疫指标(血清中IL-2、IFN-γ含量变化);无菌取脾,MTT法检测脾淋巴细胞增殖能力。
结果:
1与正常组小鼠相比,各模型组小鼠一般情况发生改变,表现出一系列的病理症状。在整个造模期间各模型小鼠活动情况明显下降,出现精神萎靡,体重增长速度减慢或体重减轻,进食饮水量减少,出现稀便,毛色失去光泽,部分小鼠出现足背水肿及关节肿大,皮肤发红等。各低免模型组相比外邪模型组,其病理症状出现的时间更早,且较为严重。其中对进食饮水量的影响以寒湿、湿热外邪较为显著,对体重的影响以寒湿外邪较为显著。
2各模型组血清中IL-2、IFN-γ的含量及脾淋巴细胞增殖能力明显低于正常组,经统计学处理,均有显著性差异(P<0.05或P<0.01)。其中各低免模型组的下降程度更大。其中以寒湿外邪对血清中IL-2、IFN-γ的含量及脾淋巴细胞增殖能力的影响相对更为显著。
结论:
1从生态医学角度提出人体既要“天人合一”又要“正气存内”,方可“邪不可干”,指出疾病的发生是机体与外界环境相关性失衡所致,是多种因素致病。
2实验显示在长期“太过”气候的影响下,会出现机体的病理状态;不同外邪因素均会引起机体免疫功能的下降,即外邪导致正气不足使机体与外界环境状态失衡而发病;外邪对免疫低下小鼠其影响更为显著;综合实验结果,在长时间风寒邪气、寒湿邪气、湿热邪气的刺激中,以寒湿邪气刺激对机体免疫功能的影响更为显著。
Objective:
This thesis,beginning with ideological theory of Ecological Medicine of Huangdi Neijing (hereinafter referred to as Neijing), discovers the traits of disease and pathogenesis caused by six pathogenic factors. Furthermore, the thesis analyzes the effect of the general conditions and immune state on normal mice and immunodeficiency mice treated by different exogenous pathogens according to experiments, looking for relativity among diseases caused by exogenous pathogen, environmental factors and the state of organism. This research prepares the academic ground and experimental reference for the research of the modern ecology etiology, which provides new thoughts and methods for diagnosis and treatment of diseases caused by exogenous pathogen.
Methods:
1 Group dispensation: 64 Kunming mice, the amount of male and female mice being equal in, are divided into 8 groups as normal group, wind-cold group, damp-cold group, damp-heat group, immunodeficiency group, wind-cold immunodeficiency group, cold-damp immunodeficiency group and damp-heat immunodeficiency group in random. Each group contains 8 mice, weighting between (20±2) g.
2 Model:
(1) The mice were put into relevant exogenous pathogenic model of wind-cold, damp-cold and damp-heat environment and treated eight hours per day for seven days. Through the observation of the mice, the author recorded the conditions of energy, activity, coat color, skin, joint and urine and stool. At the same time, taking food and water consumption as well as the weight were recorded regularly.
(2) Preparation immunodeficiency model: The normal mice were normally bred 2 days, and then the mice were given injection of cyclophosphamide(CY)80mg/kg through abdomens in each immunodeficiency model,injecting CY continuously 3 days. The rest operating procedures are same as mentioned above.
3 Detection:
(1)The mice were put into the environment of exogenous pathogen each day, observed and recorded the change of general conditions.
(2)After establishing the model of cyclophosphamide, the mice, which in the environment of exogenous seven days, were extracted samples. The specific measures include operating procedures as follows. The blood of mice was taken by removing the eyeballs. The immune indicators of mice were tested by taking serum (the change of contents of IL-2、IFN-γin serum). The spleens of mice were taken aseptically, and then the ability of lymphocyte proliferation in spleen was tested with the method of MTT.
Trial results:
1 The general conditions of the mice of wind-cold, damp-cold and damp-heat groups have changed, which shows a series of pathological symptoms when compared with normal group. During the whole model, the conditions both mental and physical of mice in each group declined that mice appeared depressed, the increase of weight has been moderate, taking food and water consumption has been reduced, looser stools, and lusterless coat color. Furthermore the part of mice developed symptoms with swollen joint, redness of the skin and even ulcerated through the skin and so forth. The pathologic symptoms appear in the immunodeficiency group, which are earlier and seriously than other groups.
2 The level of IL-2 and IFN-γin serum and the ability of lymphocyte proliferation in spleen of the mice in each model are significantly lower than that of the mice in normal group, and there is significant discrepancy in statistics (P <0.05 or P <0.01). The test indexes of immunodeficiency group were much lower than that of other groups. The results showed that all the models of exogenous pathogen as wind-cold, damp-cold and damp-heat could cause decline of the immune function of organism and diseases.
The level of IL-2 and IFN-γin serum by experiment shows that there is significant change among wind-cold group, damp-cold group and damp-heat group of immunodeficiency group, in which the test indexes of wind-cold group and damp-cold group are lower obviously with comparison among groups. The ability of lymphocyte proliferation in spleen by experiment shows that there is significant discrepancy of wind-cold group and damp-cold group among immunodeficiency group.
Conclusion:
1 Based on the theoretical research of pathogenesis of six pathogenic factors, this thesis concludes that the occurrence of diseases is mainly due to such two factors as health qi in human body and exogenous pathogen (or six pathogenic factors). The mice are growing in natural climate under normal circumstances, while the mice appear pathological symptoms of organism under influence of beyond normal range of climate. Meanwhile, with points of view of Ecological Medicine, standpoints are presented that human body requires“Correspondence between Man and Nature”and“sufficient health qi inside the body”that“the pathogenic qi have no way to invade the health body”, and the cause of diseases are produced by several factors that is led by ecological pathogen.
2 Therefore the experiment has proved that different exogenous pathogen all can cause the decline of immunity of organism that means exogenous pathogen cause weakness of body to produce disease. During the long-playing stimulate amid wind-cold pathogenic factor, damp-cold pathogenic factor and damp-heat pathogenic factor, the influence for immunity of organism by damp- cold stimulate is more distinct.
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