下丘脑—垂体—甲状腺轴细胞分子机制的季节变化与中医肾本质研究的探讨

【作者】 张明泉；

【导师】 郭霞珍；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2007， 博士

【摘要】 天人相应学说是中医理论体系的核心思想,它对中医理论的阴阳、五行、藏象、气血津液等有着重要的影响,尤其在藏象理论中,天人相应学说更是通过季节气候将五脏、六腑等与自然融为一体,形成了具有中医特色的“五脏应时”理论。然而目前对季节气候如何通过人体对五脏发挥作用的机制研究还处在初始阶段,所以如何运用现代科学技术,以中医天人相应的整体思维模式为基础,建立“五脏应时”的实验研究是亟待解决的问题。1.目的:以中医“肾应冬”为“五脏应时”研究的突破口,以下丘脑-垂体-甲状腺轴为主线,通过对其理论内涵的剖析,提出与肾实质密切相关的甲状腺轴研究的可行性思路,并通过内分泌激素水平、细胞信号转导水平的相关性实验研究,进一步证实该思路的可行性与科学性,从而为中医五脏的实质研究开辟新的方向和领域。2.方法:2.1理论研究:本文采用文献资料法和逻辑分析法,对中医“五脏应时”学说的中医理论内涵和神经内分泌免疫网络整体调节理论基础进行阐释和分析,提出中医“五脏应时”学说现代研究的可行性思路和途径,并对其实验研究的切入点进行探讨。2.2实验研究:(1)总体设计:本实验研究主要以“下丘脑-垂体-甲状腺轴”为主线,以对光强度极为敏感的松果腺为切入点,选择了在细胞信号转导途径中起重要作用的G蛋白和受体,以及内分泌系统分泌并释放的激素作为研究的对象,观察在生理状态下,大鼠冬夏季节下丘脑-垂体-甲状腺轴相应水平的G蛋白、TSH受体(MR)、TRH受体mRNA及促甲状腺激素释放激素(TRH)、促甲状腺激素刺激激素(TSH)、三碘甲状腺氨酸(T3)、四碘甲状腺原氨酸(T4)的变化。同时观察松果腺在此过程中的高位调节作用。(2)实验动物:SD大鼠,雄性,180-200 g/只。随机分成生理组、手术组、伪手术组,每组8只。实验大鼠接受自然光照,自由摄取食水,室温饲养(冬季室温为17℃±2℃,夏季室温为25℃±2℃)。在每年冬至、夏至日前一个月,手术组大鼠结扎矢状窦并摘除松果腺,伪手术组大鼠结扎矢状窦但不摘除松果腺,生理组大鼠不做任何手术。所有实验大鼠将被饲养至冬至、夏至当日晚九点,断头处死。(3)检测方法:G蛋白及TSH受体含量测定,运用Western Blot;TRH、TSH、T3、T4测定,运用放射免疫法;TRH受体mRNA,运用RT-PCR法。(4)分析方法:受体、G蛋白和TRH受体mRNA含量分析运用AlphaImager TM图像分析系统,所有数据将接受裂区设计的实验分析,该分析将运用SAS 8.02软件进行统计,并进行q检验。3.结果3.1理论研究结果(1)褪黑素在中医基础理论中发挥的作用非常关键,它是阐明中医基础理论中天人相应的整体思想的关键所在,也是研究“时脏相应”揭示五脏实质的关键所在,更是研究节律提倡因时制宜的关键所在,具有不可替代的重要地位。(2)西医的神经-内分泌-免疫网络注重机体的整体功能调节,符合中医“天人相应”的整体思想,体现了中医五脏的相生相克,制化平衡的机制。它是阐明五脏之间相互关系的物质基础,也是阐明五脏本身的物质基础。(3)以“五脏应时”为理论依据,通过“时”这个节律性变化因素研究神经-内分泌-免疫网络,既可以保证该网络体系研究的生理特性,又体现了其网络体系的整体性和特殊性,其研究方法符合中医的整体思想和以生理推断五脏实质的研究思路。3.2实验研究结果3.2.1季节因素对下丘脑-垂体-甲状腺轴的影响(1)血清内分泌激素:T3(P<0.01)、T4(P<0.05)、TRH(P<0.05)三项指标冬夏比较:冬高夏低,差异显著;TSH含量冬夏比较没有显著性差异。(2)受体水平:甲状腺TSH受体含量冬夏比较:冬高夏低,差异显著,P<0.05。(3)基因水平:垂体TRH受体mRNA含量冬夏比较:冬高夏低,差异显著,P<0.05。(4)甲状腺轴G蛋白:下丘脑Gi、垂体Gq两项指标冬夏比较:冬高夏低(P<0.05),差异显著;而甲状腺Gi和甲状腺Gs冬夏比较没有显著差异。3.2.2手术因素对下丘脑-垂体-甲状腺轴的影响(1)血清内分泌激素:SD大鼠血清T4在冬至生理组和冬至手术组之间比较有显著性差异,P<0.05;T3、TSH、TRH在生理组、手术组及伪手术组间比较没有显著性差异。(2)受体水平:TSH受体含量在在生理组、手术组及伪手术组间比较没有显著性差异。(3)基因水平:TRH受体mRNA含量在生理组和手术组之间比较有显著性差异,P<0.05。(4)甲状腺轴G蛋白:下丘脑的Gi、甲状腺的Gs蛋白在生理组和手术组之间比较都有显著性差异,P<0.05;垂体的Gq蛋白在冬至生理组和冬至手术组之间比较有显著性差异,P<0.05;甲状腺的Gi蛋白在在生理组、手术组及伪手术组间比较没有显著性差异。3.2.3手术与季节的交互作用。(1)血清内分泌激素:SD大鼠血清T3(P<0.05)、T4(P<0.05)、TSH(P<0.01)三项指标各自存在手术与季节的交互效应,也就是说季节因素产生的差别,可因手术施加的程度而异。而血清TRH没有手术与季节的交互效应。(2)受体水平:SD大鼠甲状腺TSH受体含量没有手术与季节的交互效应。(3)基因水平:SD大鼠垂体TRH受体mRNA含量没有手术与季节的交互效应。(4)甲状腺轴G蛋白:SD大鼠垂体Gq蛋白的含量存在手术与季节交互效应(P<0.05)。而下丘脑Gi、甲状腺Gs和Gi三项指标各自没有手术与季节的交互效应。4.结论(1)中医“肾应冬”调控机制与季节因素密切相关。(2)中医“肾应冬”调控机制与松果体密切相关。(3)从“五脏应时”角度研究中医脏象实质的思路是可行的。(4)本研究首次以季节变化为因素,以下丘脑-垂体-甲状腺轴为主线,从整体角度深入研究细胞信号转导机制,并通过实验证实中医“肾应冬”的规律与下丘脑-垂体-甲状腺轴整体调节机制下的细胞信号转导网络被激活密切相关。(5)本研究首次发现,手术摘除松果体对下丘脑-垂体-甲状腺轴的季节性变化规律有拮抗作用,也就是说褪黑素对甲状腺轴的影响在季节方面具有双重性。进而本文从细胞信号转导水平对该双重性进行了分析和论证。其实验意义是具有开创性的。更多还原

【Abstract】 Correspondence between Man and Universe is the core of TCM theory. It has great influence on the following theories as Yin-Yang ,Five-Elements, State of viscera, Qi, Blood and Body Fluid et al.Especially on the theory about State of viscera, the theory of Correspondence between Man and Universe, binding with Five-Zang Organs, Six Fu Organs and the season, becomes the“Five-Zang proper to the season”theory with the character of TCM. At present, however, the study on the mechanism of how“Five-Zang proper to the season”functions on Five-Zang Organs is at the initial phase. As a result, how to employ modern science to establish the experimental study on“Five-Zang proper to the season”, basing on the integrative thinking of Correspondence between Man and Universe, becomes the urgent problem.1. ObjectiveTaking the idea of“kidney corresponds with winter”in TCM as a breaking point to“Five-Zang proper to the season”theory, and taking the hypothalamic-pituitary-thyroid axis as the main part, we propose a feasible way to study on thyroid axis, which is closely related with renal essence, through deep analysis on its theory. Furthermore, we have performed the experimental research on the level of endocrine hormone and Cells signal transduction to prove their scientific and feasible value. Thus we suggest that there is a new direction and field for the study on the essence of Five-Zang.2. Methods2.1 Theoretical methods:The methods of documentary materials and logical analysis are used to explain and analyze the theoretical connotation of“Five-Zang proper to the season”and the overall regulatory control of neuroendocrine-immune network. The present paper puts forward a feasible way for the present study on the Zang-Xiang theory of TCM and discusses the breaking point to its experimental study.2.2 Experimental methods2.2.1 Overall designThe present experiment takes hypothalamic-pituitary-thyroid axis and pineal gland as the main clue.Taking the light-sensitive pineal body as the key point, the present sutdy selects G protein and receptors, which is of importance in the pathway of cell signal transduction, and endocrine hormone as the objects, the present author tries to oberve, with the changes of winter and summer,the changes in rats under physiological condition, changes of G protein, TSH receptor, T3, T4 , TSH, TRH, TRH Receptor mRNA of hypothalamic-pituitary- thyroid axis. Meanwhile, the regulation role of pineal gland at a high level during this process is also observed. 2.2.2 Experiment animalsSprague-Dawley rats (male), weighted 180-200 gram each, are randomly divided into three groups as controlled group, operation group, and false operation group. There are eight rats in every group.All rats will receive natural light, ate and drank freely, and will be controlled under certain room temperature(room temperature is 17℃±2℃at winter, and it is25℃±2℃at summer).One month before Winter Solstice and one month before Summer Solstice every year, the rats of operation group will ligate their arrow veins and remove their pineal glands,while the rats of false operation group will only ligate their arrow veins,but not have their pineal glands cut down,and the rats of controlled group will have no operation at all. All the rats will be raised till Winter Solstice and Summer Solstice, on that day at nine o’clock at night they will be beheaded.2.2.3 Test methodsTo assay the content of G protein and TSH receptor, Western Blot is engaged; to assay the content of TRH Receptor mRNA, RT-PCR is engaged; and to assay the content of T3, T4,TSH,TRH, radio-immunity is engaged.2.2.4 Analysis methodsImage analysis software of AlphaImager TM is engaged for G protein TSH receptor and TRH Receptor mRNA. All date will employ SAS 8.02 software to perform split-plot design analysis and receive q-test.3. Results3.1 Results of theoretical study(1) Melatonin plays a very important role in the theories of TCM. It forms the core to explain the integrative idea of the correspondence between Man and Universe. Meanwhile, it is also the key point in explaining the essence of Five-Zang in the study of“Five-Zang proper to the season”. Melatonin is also the key point to study rhythem and to promote climate concerned therapy, thus the significance of melatonin in the theories of TCM cannot be replaced.(2) Neuroendocrine-Immune Network in modern medecine pays importance to the regulation of integrative functions of mechanisms, and this idea agrees with the integrative idea of correspondence between Man and Universe in TCM, therefore this idea embodies the mechanism of inter-promotion, inter-restriction,and interaction of Five-Zang in TCM. Neuroendocrine-Immune Network forms the material basis for the explanation of the interrelation among Five-Zang, as well as for each Zang.(3) To study Neuroendocrine-Immune Network (NEI), the present author makes“Five-Zang proper to season”as the theoretical basis, and chooses seasons as a variable factor, thus it garantees the physiological nature of the study of NEI and also embodies the integrative and special features of NEI. This study method agrees with the integrative idea of TCM, and it agrees with the idea of deducing the essence of Five-Zang from physiology as well.3.2 Results of experiments3.2.1 The effects of seasons (Winter Solstice and Summer Solstice) on hypothalamic-pituitary-thyroid axis(1)blood serum endocrine hormone: T3(P<0.01), T4(P<0.05),TRH(P<0.05)in blood serum endocrine hormone are observed to have significant difference between winter and summer (they are higher in winter and lower in summer). However, there is not much difference in the contents of TSH.(2)on the level of receptor: the contrast about the contents of thyroid in TSH receptor shows that the contents is relatively higher in winter while lower in summer,(there is significant difference in the content, P<0.05).(3)on the level of genes: the contrast about the contents of pituitary in TRH Receptor mRNA shows that the contents is relatively higher in winter while lower in summer,(there is significant difference in the content, P<0.05).(4) thyroid axis G-protein: the contrast of hypothalamusGi and pituitaryGq in winter and summer: there are significant difference between winter and summer (they are higher in winter and lower in summer, P<0.05), However, there is not much difference in Gi and Gs of thyroid between winter and summer.3.2.2 The influence of operation on hypothalamic-pituitary- thyroid axis(1)blood serum endocrine hormone:there is significant difference in T4 between the controlled group in winter and the operation group in summer(P<0.05), while for the following data as T3,TSH,TRH, there is not much difference among the controlled group,operation group and false-operation group.(2)on the level of receptor: there is not much difference among the controlled group,operation group and false-operation group in the content of TSH.(3)on the level of genes: there is much difference between the controlled group and operation group in mRNA of TRH-Receptor.(4)thyroid axis G-protein: there is significant difference in Gi of hypothalamic and in Gs of thyroid between the controlled group and the operation group(P<0.05);there is much significant difference in Gq of pituitary between the controlled group in winter and the operation group in summer(P<0.05); the following data as Gi in thyroid, there is not much difference among the controlled group,operation group and false-operation group.3.2.3 corelation of operation and season(1)blood serum endocrine hormone: there is corelation between operation and season for the respective factor of blood serum as T3(P<0.05), T4(P<0.05),TSH(P<0.01)of SD rats. In another word, the difference of season alters with the degree of operation. There is no correlation between operation and season in TRH of blood serum.(2)on the level of receptor: there is no corelation between operation and season in the content of thyroid Tsh-Receptor of SD rats. (3)on the level of genes: there is no corelation between operation and season in the content of mRNA of pituitary TRH of SD rats.(4)thyroid axis G-protein: there is corelation between operation and season in the content of Gq of SD rats. There is no corelation between operation and season in the respective factor as Gi of hypothalamus, Gs and Gi of thyroid.4. Conclusions(1) There is close relationship between the modulating mechanism of“kidney proper to winter”and season.(2) There is close relationship between the modulating mechanism of“kidney proper to winter”and pineal gland.(3) It is feasible to study the essence of state of viscera in TCM from the point of“Five-Zang proper to season”.(4) It is the present author who,for the first time, takes the changes of seasons as factors and takes hypothalamic-pituitary-thyroid axis and pineal gland as the main clue to make a integerative and deeper study about the mechanism of cell signal transduction. It is also the present author who, for the first time, proves that the rule of“kidney proper to winter”of TCM is closely related with the irrigation of cell-signal transduction (under the integrative control of hypothalamic-pituitary- thyroid axis).(5) In present study, the author discovers that the result of removal of pineal gland will have antagonistic effect on the hypothalamic-pituitary- thyroid axis, which is influenced by the changes of seasons. In another word, melatonin has the feature of dualism on the thyroid axis, which is influenced by the changes of seasons. In addition, the present paper evaluates and analyzes the dualism on the level of cell- signal transduction. The significance of the present experiment lies in the new ideas it brought forth.更多还原