【作者】 杨裕华；

【导师】 李震；

【作者基本信息】 山东中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 目的:本研究旨在利用基因芯片技术探讨“劳倦过度、房室不节”肾阳虚模型小鼠及其补肾中药金匮肾气丸、右归丸治疗后脑基因表达谱的改变,“以药反证”,并在分子水平上研究药物的作用机理,同时还比较二方剂作用的基因变化。方法:对照组、造模组、金匮肾气丸组和右归丸组,各组雄性小鼠随机为10只、15只、10只和10只。四组均予正常喂养,对照组和造模组每天灌胃蒸馏水0.5mL/只,二治疗组每天分别灌胃金匮肾气丸混悬液0.5mL/只和右归丸混悬液0.5mL/只,造模组和二治疗组均采用雄雌鼠比例1:6同笼喂养和雄鼠每日游泳至无力下沉共4周,以诱导产生“劳倦过度、房室不节”肾阳虚证。用36K mouse genome array鼠脑芯片检测正常对照组和肾阳虚模型组鼠脑基因,并以二者荧光信号相对强度比值≥2和≤0.5筛选差异显著基因,并进一步查阅北京博奥生物分子注释系统,进行基因功能归类。为进一步验证芯片所检测的差异基因,还应用Real-Time PCR对部分差异显著基因进行检测验证。结果:造模组诱导成功“劳倦过度、房室不节”肾阳虚鼠模型,同时通过基因芯片检测,分别绘制了造模组/对照组与二治疗组/造模组基因表达谱的散点图。造模组/对照组共筛选出186个差异表达基因,其中上调基因150个,下调基因36个。金匮肾气丸组/造模组筛选出299个差异表达基因,其中上调基因114个,下调基因185个。右归丸组/造模组筛选出155个差异表达基因,其中上调基因64个,下调基因91个。二治疗组/造模组基因均上调的基因24个,分别占各组上调基因的21.05%和37.50%,其主要是与神经传递/信号转导、转录/翻译、激素、细胞周期和代谢相关的基因;二治疗组均下调的有35个基因,分别占各组下调基因的18.92%和38.46%,其主要是与转录/翻译、神经传递/信号转导、炎症/免疫、代谢以及影响多巴胺生成的限速酶有关基因。造模组/对照组上调基因而治疗组/造模组基因下调的有23个基因,其主要是与炎症/免疫、神经传递/信号转导等相关基因;造模组/对照组下调基因而治疗组/造模组基因上调的基因有6个,其主要是与细胞周期/细胞结构、神经传递/信号转导、转录等有关基因。结论:造模组上调基因主要涉及与炎症/免疫、氧化应激等有关基因,使得炎症/免疫和氧化反应增强,创伤、损伤反应和凝血机制增强,这些都是机体的保护性反应所致,还有多巴胺神经介质分泌减少引起多巴胺能神经紊乱。下调基因主要与激素(特别是黑色素浓集素和性激素)、细胞周期、信号转导/神经传递、代谢(特别是蛋白质代谢)和生长发育(特别是脑发育和精子发育、受精等)相关基因。金匮肾气丸、右归丸可使肾阳虚小鼠模型显著下调的激素、黑色素浓集素显著上调并促进细胞增殖,从而在基因水平上探讨了金匮肾气丸和右归丸的药物作用机理及其差别。更多还原

【Abstract】 Objective: To inquire into the cerebral gene change of the model mouse with kidney-yang deficiency caused by excessive physical and sexual activities and its variation tested by means of the gene microarry technology, its improved syndrome change due to using drugs of efficacy such as Jinkuishenqiwan and Youguiwan which reinforcing the kidney-yang according to traditional Chinese medicine, the mechanism of the medicine, and a comparison of the genetic change between the two medicine on a molecular level.Methods: There were four groups randomly: the control group which has 10 male mice, the model group which has 15 male mice, the Jinkuishenqiwan treatment group which has 10 male mice and the Youguiwan group which has 10 male mice. All of them were fed normally, and perfused with 0.5 mL of distill water for each mouse in the control group and model group, and with 0.5 mL suspension of the drugs for each in the treatment groups every day. The experimental mice in the model group and the treatment groups were kept with six female mice each in the same cage, and all of the male mice swam until they submerged and scooped up from water everyday, which lasted four weeks. The mice might induce the kidney-yang deficiency because of excessive physical and sexual activities. The brain gene of the mice between control group, model group and treatment groups was detected with a mouse brain gene chip of 36K Mouse genome array made by CapitalBio Corp. Beijing, China, which screened the differentially expressed genes according to the ratio equal to or above 2 and equal to or below 0.5 with the related fluorescent intensity comparing with the two groups’, which could be further classified in the light of gene function using Molecule Annotation System (MAS) created by CapitalBio Corp. Beijing, China, and with related reference material.Results: The animal model of kidney-yang deficiency in the model group was induced by way of excessive physical and sexual activities successfully, and the scatter plots of expressed genes from the three groups were contrastingly drawn. Between the model and control groups one hundred and eighty-six differentially expressed genes were screened, including 150 up-regulated genes and 36 down-regulated genes. Between the Jinkuishenqiwan treatment and model groups two hundred and ninety-nine differentially expressed genes were screened, among which there were 114 up-regulated genes and 185 down-regulated genes. Between the Youguiwan treatment and model groups one hundred and fifty-five differentially expressed genes were screened, among which there were 64 up-regulated genes and 91 down-regulated genes. The twenty-four genes among up-regulated genes in the two treatment groups versus model group, account for 21.05 percent and 37.5 percent in each treatment group respectively, which are mainly the genes related to neurotransmissions/signal transduction, transcription/translation, cellular cycle, hormone and metabolism. However, down-regulated genes in the treatment group versus model group were 35, accounting for 18.92 percent and 38.46 percent in each treatment group respectively, chiefly including the genes association with inflammation/immunization, neurotransmissions/signal transduction. transcription/translation, metabolism and limiting velocity enzyme of dopamine. There were twenty three genes among the up-regulated genes in the model group versus control group but down-regulated in the treatment group versus model group, mainly involving the related genes of inflammation/immunization and neurotransmissions/signal transduction. There were six genes among the down-regulated genes in the model group versus control group but up-regulated in the treatment group versus model group, mainly including the related genes of cellular cycle and structure, neurotransmissions/signal transduction and transcription.Conclusion: The up-regulated genes in the model group mainly involed the genes related to inflammation/immunization, oxidative stress and so on, which made enhancing response to inflammation/immunization and oxidation, wound and impairment, also to agglutination. The phenomenon by above-mentioned is caused by protective response of the organism. In addition, secretion of decreased dopaminergic transmitter can cause dopaminergic disorder. The down-regulated genes included hormone (especially melanin-concentrating hormone (MCH), gonadal hormone, cell cycle, signal transduction/neurotransmission, metabolism (particularly proteometabism), growth and development, (especially cerebral development, sperm development fertization etc). Jikuishenqiwan and Youguiwan may make it markedly up-regulated to notably down-regulated genes of hormone and melanin-concentrating hormone(MCH) for model of mice of kidney-yang deficiency, and promote cellular proliferation, which can inquire into effect mechanism of the two drugs and their differences in genetic level at the same time.更多还原