中医“肾应冬”调控机制与细胞信号转导相关性的研究

【作者】 刘晓燕；

【导师】 刘燕池；

【作者基本信息】 北京中医药大学 ， 中医基础， 2004， 博士

【摘要】 脏象学说是中医理论体系的核心内容,它贯穿于中医全部医学理论之中,并起着指导作用。所以对脏象实质的现代研究对中医理论体系的发展具有至关重要的意义。然而,目前在对脏象实质的现代研究中,如何恰当地运用现代生命科学的研究成果,建立符合中医学本身理论思维模式和中医脏象特色的可行性研究思路和途径是亟待解决的问题。1.目的: 以中医“肾应冬”调控机制为研究对象,通过对其理论内涵的剖析和前期实验结果的总结,提出中医脏象实质现代研究的可行性思路。并通过“肾应冬”调控机制与细胞信号转导相关性的实验研究进一步证实该思路的可行性与科学性,从而为中医脏象学说的实证研究开辟新的方向和领域。2.方法:2.1 理论研究:采用文献学、逻辑分析等方法,对中医“肾应冬”调控机制的中医理论内涵和现代细胞生物学整体调节理论基础,及前期的实验结果进行阐释和分析,提出中医脏象实质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:本实验研究主要以与“肾应冬,主生殖”相关的性腺轴和对光节律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 蛋白和受体分子,以及在细胞信号转导末端的基因表达作为研究的对象。观察在生理状态下,大鼠冬夏季节下丘脑-垂体-性腺轴相应水平的 G 蛋白、褪黑素受体(MR)、FSH 受体及睾丸组织的基因表达的变化。同时观察松果腺在此过程中的高位调节作用。另外,本研究还对性腺轴高位调节激素,促性腺激素释放激素(GnRH)的冬夏季节变化进行了观察。(2)实验动物:SD 大鼠,雄性,180-200 g/只。随机分成生理组,手术组(结扎矢静脉并摘除松果腺组),伪手术组(结扎矢静脉但不摘除松果腺组)。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、夏至日前一个月对实验大鼠进行松果腺摘除手术和伪手术,术后饲养至冬至日、夏至日,断头处死。(3)检测方法:G 蛋白含量测定,运用 Western Blot;褪黑素受体和 FSH 受体测定,运用放射配体饱和法;睾丸组织基因差异显示,运用 DDRT-PCR 法;GnRH 含量测定,运用放射免疫法。(4)分析方法:受体分析运用 Prism 3.02 分析软件,其它检测与分析数据运用 SPSS10.0 分析软件进行 T 检验或 F 检验等统计学检验方法。3.结果3.1 理论研究结果 ① 本项研究立足于从人体主体性开放的整体调控观、自组织的适应性调节规律、<WP=5>4 中医“肾应冬”调控机制与细胞信号转导相关性的研究时间结构相关等创新性认识对“肾应冬”调控理论的现代内涵进行深入的剖析,发现中医学的五脏在与“时”相应的适应性调控过程中,不仅能在生理状态下体现出中医五脏的天人相应、整体调控特点,而且还能充分展现五脏的时空特性。在此启发下,本研究首次提出了:从“天人相应”脏腑应时适应性调控的角度,来实证研究中医脏象的思路。 ② 本研究通过对“肾应冬”调控机制的现代细胞生物学整体调节理论基础的分析,发现在理论上“肾应冬”调控机制与细胞信号转导存在相关性,并提出了从细胞信号转导的角度来研究“肾应冬”的调控机制,符合中医学对五脏功能调控实质的认识。 ③ 本研究提出了中医“肾应冬”调控机制的工作假说,即:中医“肾应冬”的调控机制是,机体在冬季受到相应环境信息(如光照等)的刺激后,激活了细胞信息转导的载体——细胞信号转导系统,并将信号传导于神经内分泌免疫网络和体内器官,从而形成了一系列与冬季相应的有规律的综合生理效应。这些生理效应符合中医“肾者,主蛰,封藏之本,精之处也……通于冬气”的论述。3.2 实验研究结果3.2.1 冬夏季节对机体细胞信号转导物质及 GnRH 的影响 在生理条件下,冬夏季节的变化对体内性腺轴相关的细胞信号物质及激素均有显著影响,表现在: ①下丘脑 G 蛋白四个亚型中,Gq(P<0.05)、Go(P<0.05)含量均呈现明显的夏高冬低,Gs、Gi 无明显差异。 ②褪黑素受体: 下丘脑(P<0.05)和垂体(P<0.05)Bmax 值均表现为冬高夏低。下丘脑(P<0.05)和垂体(P<0.01)KD 值均表现为冬高夏低。 ③睾丸 FSH 受体: Bmax(P<0.01)和 KD(P<0.01)值均表现为夏高冬低。 ④睾丸组织基因差异显示: 夏季有表达而冬季无表达的基因有 2 个。夏季无表达而冬季有表达的基因有 9 个。夏季表达强冬季表达弱的基因有 2 个。夏季表达弱而冬季表达强的基因有 10 个。 ⑤ GnRH: 血浆 GnRH 含量表现为夏高冬低(P<0.05)。下丘脑中 GnRH 含量冬夏没有显著性差异(P>0.05)。3.2.2 松果腺对细胞信号转导物质及 GnRH 的季节影响 在摘除松果腺的情况下,体内性腺轴相关的细胞信号物质及激素水平的正常节律均会发生变化。表现在: ① 下丘脑 G 蛋白的四个亚型,手术组与生理组相比均有不同程度的改变(但没有统计学意义,P>0.05)。Gq、Go 的正常冬夏季节差异消失。 ② 褪黑素受体:冬季手术组下丘脑(P<0.01)、垂体(P<0.01)Bmax 值都明显高于生理组,夏季手术组下丘脑(P<0.01) Bmax 值明显高于生理组。冬、夏手术组下丘脑(P<0.01)更多还原

【Abstract】 Zang-Xiang theory is the core of TCM theory. It impenetrates all medical theory of TCM ,with the effect of guidance. So it is of fairly important significance of modern research on theZang-Xiang theory to the development of TCM theory. However, what the urgent problem ofstudying on the Zang-Xiang is how to make good us of the achievements of modern scienceof life, and how to put forward feasible research way that is in accordance with characteristicsof Zang-Xiang in TCM.1. ObjectiveThis study tends to propose feasible way to the study of the Zang-Xiang theory of TCM by acomprehensive analysis of the connotation of the theory and the summary of the results ofpreliminary experiment, with the modulating mechanism of ‘kidney corresponds withwinter’ as object. And further evidence of the feasibility of the way would be provided by theexperimental study on the relationship between the modulating mechanism of ‘kidneycorresponds with winter’ and cell signal transduction, which is in order to break a new pathfor the study on the zang-xiang theory of TCM.2. Methods2.1 Theoretical methods:Literature survey, logic analysis and other methods were used to analyze the modernconnotation of modulating mechanism on the ‘kidney corresponds with winter’ of TCM andresults of the preliminary experiment, which is in order to propose feasible way to study onthe Zang-Xiang theory of TCM and discuss the key point to experiment on it.2.2 Experimental methods2.2.1 Collectivity designThe experiment study makes gonad axis and pineal gland as the key point. G protein andreceptors, which is of importance in the pathway of cell signal transduction, and geneexpression that is at the end of cell signal transduction were selected as the object of thisstudy. Under physiological condition, changes of G protein, melatonin receptor, FSH receptorof hypothalamic-pituitary- gonad axis and the gene differential expression of testicle in rats,corresponding to winter and summer, were investigated. The regulation role of pineal gland ata high level during this process was also observed. Moreover, the content of hypothalamusand plasma GnRH of rats during winter and summer were observed, too.2.2.2 Experiment animalSprague-Dawley male rats, weighted 180-200 gram, were randomly divided into groups ascontrol, model (ligation of arrow vein and removal of pineal gland), false operation (ligationof arrow vein without removal of pineal gland) . natural light, ate and drank freely, roomtemperature(room temperature was17℃±2℃ at winter, it was25℃±2℃ at summer).<WP=8>英文摘要 7The false operation and the operation of removal pineal gland were carried out at 1 monthbefore the Winter Solstice and the Summer Solstice each year, rats were bred after theoperation and were decollated at night on the Winter Solstice and the Summer Solstice.2.2.3 Test methodsFor content of G protein, Western Blot was engaged; for melatonin receptor, FSH receptor,radioligand saturation was engaged; for differential expression of the gene of testicle,DDRT-PCR was engaged; for GnRH content, radio-immunity was engaged.2.2.4 Analysis methodsPrism 3.02 for analysis was engaged for receptor analysis. And statistical test methods, suchas T test, F test et al. was engaged for other measurements and data analysis.3. Results3.1 Results of theoretical study① In the study, the modern connation of ‘kidney corresponds with winter’ modulatingmechanism was analyzed by the view of integrated modulating mechanism of human body,self-organization, time structure, et al.. It was found that during the process of adaptableregulation of the body corresponding with seasons, the characteristic of Five Zang organs inTCM, such as integrated modulation and time characteristic, can be sufficiently exhibited.With the elicitation, it was proposed for the first time in this study that what is the feasibleway for rese更多还原