以葛根素为探针解析SD大鼠时辰节律的研究

【作者】 戴进；

【导师】 赵琰； 王庆国；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2012， 硕士

【摘要】 在生命过程中,从分子到细胞、从个体到群体都具有明显的周期往复的现象。已有研究显示,人类多种宏观生命活动以及微观的生理指标,如内分泌激素、微量元素等都具有的规律性周期活动。随着医学科学的发展,人们发现许多药物的疗效除了与病人所患疾病及药物本身特性、用量大小、用药途径等因素有关外,还与机体节律性密切相关。因此,适应生物节律制定合理的给药方案,对提高药物疗效、降低毒副作用、具有重要的临床意义与价值。目前,在对药物作用的时间节律和药物对机体生物节律的影响,及临床治疗最佳用药时间的研究中,科研方法主要局限于通过检测血药浓度等内源性理化指标来获得药物体内代谢及分布的时辰节律,但是对外源性物质作用靶点的时辰节律变化尚缺乏有效的研究手段。随着现代科学技术的飞速发展,单克隆抗体免疫分析技术逐渐成熟,因其具有特异性强、灵敏度高、可靠安全、快捷方便等优点在医学临床检验和基础研究中获得广泛应用。本团队基于自主制备的中药小分子葛根素与栀子苷的单克隆抗体,采用免疫分析法可以特异灵敏的检测单味药和复方中葛根素与栀子苷在体内的代谢分布,其中葛根素灵敏度可达到匹克级。因此以中药小分子物质为探针,通过检测不同时辰其在体内靶器官分布情况。以此作为一种新的思路与手段来证明时辰节律的本质内涵。从而为达到理想的治疗目的提供科学依据。目的：本论文旨在通过应用葛根素单克隆抗体为探针,解析葛根素在SD大鼠体内时辰节律的分布趋势,为基于人体节律的时间医学研究提供新的思路与方法。方法：1分组和给药：将25只SD大鼠随机分成8：00组、12：00组、16：00组、20：00组、24：00组,实验前各组动物动物禁食8小时,自由饮水。在设定的各个时间点上对各组动物按20mg/kg给药剂量腹腔注射给药。2取材一小时后腹主动脉取血,处死动物,立即取心、肝、脾、肺、肾和脑,冰上对全脑分离出皮层、海马、间脑、延髓、小脑、松果体。所有组织于-40℃冷冻保存。3血液和组织前处理方法血液常温放置2h,4℃冰箱放置过夜,3000r/min离心取上清。皮层、小脑、间脑、海马、延髓、松果体等容易破碎的、重量小的组织称重,按100mg组织：1mlPBS的比例加入PBS溶液,冰浴条件下200W功率超声波粉碎匀浆15S；其余组织称取200mg,剪碎后按100mg组织：1m1PBS的比例加入PBS溶液,冰浴条件下超声破碎仪200W功率超声波粉碎匀浆15S,匀浆液于15000r／min、4℃条件下离心30min,取上清分装,于-40℃冷冻保存。结果：(1)葛根素在SD大鼠血液中其峰值相位为20：00时,24：00时浓度较低,其谷值相位为8：00,峰谷值之间相位相差12小时(p<0.05),因此周期为为近似昼夜节律。(2)葛根素在SD大鼠肾脏、肺脏、肝脏、脾脏中的分布趋势其峰值出现在12：00,而心脏中的分布趋势峰值出现在12：00,这种分布趋势的差异提示葛根素不同的靶器官具有不同的时辰节律。(3)葛根素8：00时在SD大鼠主要脏器中的药物分布趋势呈肝<脾<肺<心<肾。在12：00时在SD大鼠主要脏器中的药物分布趋势呈脾<心<肺<肝<肾。在16：00时在SD大鼠主要脏器中的药物分布趋势呈脾<肺<肝<心<肾。在20：00时在SD大鼠主要脏器中的药物分布趋势呈脾<肝<肺<心<肾。在24：00时在SD大鼠主要脏器中的药物分布趋势呈脾<肝<肺<心<肾。这种分布趋势的差异提示不同时辰下葛根素在SD大鼠主要脏器中的分布呈不同的趋势。(4)葛根素在脑部不同分区皮层、松果体、间脑、小脑、延髓、海马中的分布趋势呈近日节律的趋势,然而其相位峰值的时间却并不一致。(5)葛根素在SD大鼠不同脑区中的药物分布趋势8：00时呈松果体<间脑<延髓<海马<皮层<小脑,12：00时呈松果体<海马<间脑<皮层<延髓<小脑,16：00时呈松果体<间脑<延髓<海马<皮层<小脑,20：00呈松果体<海马<延髓<皮层<间脑<小脑,0：00时呈松果体<间脑<延髓<皮层<海马<小脑。这种分布趋势的差异提示不同时辰下葛根素在SD大鼠不同脑区中的分布亦呈不同的趋势。结论：本实验的结果提示利用葛根素单克隆抗体El isa方法,可以观察到葛根素在SD大鼠内分布趋势具有时辰节律。不同的脏器时辰节律的相位不完全一致。因此,葛根素单克隆抗体可以做为更深刻解析中医学时辰节律实质与科学内涵的探针。更多还原

【Abstract】 During the span of human being, there are the recycling circumstances existing in organs, from molecules to cells, from organisms to colonies; however, among those, life activities, like growth and reproduction, and even some more subtle physical indicators, like endocrine hormones and trace elements, are the most well-known. As the development of medical science, we have find out that the therapeutic effects are closely related to the patient’s illness, and the drug’s nature, dose, taking method, but also the rhythm of organism. Therefore, it is crucial and valuable to apply biological rhythm to prescribe in enhancing therapeutic effects and reducing side effects. However, nowadays our research in the time rhythm of drug intervention and in the influence of drug on biological rhythm are mainly limited in physical and chemical indicators and plasma concentration detection. There is still lack of an effective studying means in the rhythm change of the therapeutic effect on targets.As the high-speed development of modern science, the monoclonal antibody immunoassay technology has become maturer and maturer, which has merits in specificity, sensitivity, reliability and convenience. Our researching team utilize small molecules of TCM puerarin, extracting in immunosorbent assay, and the monoclonal antibody of geniposide to detect the metabolism distribution of puerarin and geniposide, with a sensitivity reaching pg-level. Therefore, we can demonstrate the nature of time rhythm by a updated approach of using small molecules as the probe to detect their distribution in target organs in different hours in order to provide the scientific basis for reaching ideal treatment objectiveObjective:This paper aims to provide a new approach in researching the rhythm of human body in time medical science by illustrating the distribution tendency of puerarin monoclonal antibody as the probe in the SD mouses in the double-hour rhythm.Methods:Firstly, divide25SD rats into5groups at random. There are:group eight o’clock, group twelve o’clock, group sixteen o’clock, group twenty o’clock, and group twenty-four group. Then inject each mouse20mgl/kg puerarin injection from abdominal cavity. One hour after the injection, detect the distribution tendency of puerarin in the cerebral cortex, diencephalon, hippocampus, cerebellar, oblongata, pineal gland, liver, heart, kidney, lung, spleen and blood of the SD mouse.Results:(1) The density of puerarin in the blood of SD rats reaches its peak at20o’clock at night. The density is relatively lower at8o’clock, reaching its bottom. There are twelve hours between the peak and the bottom, with a cycle time of24±4hours, which is approximate to the day-and-night rhythm.(2) The density of puerarin in the kidney, lung, liver and spleen of SD mouse reaches its peak at12o’clock.But that in the heart of it reaches its bottom at12o’clock, which is resting and calm period. This difference in distribution tendency indicates the different target organs are different in time rhythm.(3) At8o’clock, the distribution tendency of puerarin in the major organs in SD mouse presents as:liver<spleen<lung<heart<kidney. At12o’clock, that presents as:spleen<heart<lung<liver<kidney. At16o’clock, that presents as:spleen<lung<liver<heart<kidney. At20o’clock, that presents as: spleen<liver<lung<heart<kidney. At24o’clock, that presents as:spleen<liver<lung<heart<kidney. This difference in distribution tendency indicates the different time are in different distribution tendency.(4) The distribution tendency in the different zoning cortex, pineal gland, diencephalon, cerebellar, medulla and hippocampus are in the similar tendency of the daytime, but never related to their phrase.(5) At8o’clock, the distribution tendency of puerarin in the different areas in the head of SD mouse presents as:pineal gland<diencephalon<medulla<hippocampus<cortex<cerebellar. At12o’clock, that presents as: pineal gland<hippocampus<diencephalon<cortex<medulla<cerebellar. At16o’clock, that presents as:pineal gland<diencephalon<medulla<hippocampus<cortex<cerebellar. At20o’clock, that presents as:pineal gland<hippocampus<medulla<cortex<diencephalon <cerebellar. At24o’clock, that presents as:pineal gland<diencephalon<medulla<cortex<hippocampus<cerebellar. This difference in distribution tendency indicates the different areas in the head of SD mouse are in different distribution tendency in different time.Conclusion:The results of this experiment indicates that we can detect the influence of double-hour rhythm on the distribution tendency of puerarin in SD mouses by using the approach of puerarin-monoclonal-antibody-Elisa. Therefore, the method of puerarin monoclonal antibody can be used as probe for more profound research in the nature and scientific connotation of TMC double-hour rhythm.更多还原