血瘀体质理论及其外周血基因表达谱的研究

【作者】 田栓磊；

【导师】 王琦；

【作者基本信息】 北京中医药大学 ， 中医基础理论， 2009， 博士

【摘要】 目的挖掘传统中医理论中血瘀体质的相关内容,总结近现代研究成果,补充中医血瘀体质的相关理论;检测血瘀体质的外周血基因表达谱,探索其基因表达特征,为防治血瘀体质相关疾病提供依据。方法采用传统文献梳理与现代文献分析的方法,进行中医血瘀体质理论研究。在临床流行病学调查的基础上,选择13例典型血瘀体质和30例典型平和体质受试对象,抽取空腹静脉血各10ml,分离白细胞,提取总RNA,纯化后进行单轮扩增,生物素标记cRNA,应用Illumina公司的Sentrix(?)Human-6 Expression BeadChip芯片检测基因表达谱。以平和体质作为对照,得出血瘀体质的差异表达基因,对差异表达基因进行GO注释和pathway分析,并对差异表达基因的生物学功能进行评价,从基因水平阐明血瘀体质成因、特征及其发病倾向性的机制。结果理论研究发现血瘀体质的形成受先后天多种因素的影响,多因先天遗传、年老、性别、跌扑损伤、内伤七情、饮食不节、劳逸过度以及久病等因素而形成;基因研究发现,当P<0.05时,血瘀体质与平和体质表达差异基因共592个位点,其中上调基因167个位点,下调425个位点;当P<0.001时,血瘀体质与平和体质表达差异基因共有96个位点,其中上调基因24个位点,下调基因72个位点。与平和体质相比,血瘀体质者血小板易凝集,易患缺血性疾病和肿瘤类疾病;血瘀体质者参与蛋白合成、核酸合成、类固醇激素合成等生物合成功能的基因表达下调,说明血瘀体质者生物合成与代谢功能有下降的趋势;血瘀体质者与基因转录相关的基因表达下调,说明血瘀体质者在遗传信息传递方面的功能可能呈现下降状态;而血瘀体质者部分与生长发育相关的基因表达下调,以及与二氢嘧啶脱氢酶、人甲基转移酶、蛋白精氨酸甲基转移酶、UDP葡萄糖焦磷酸化酶、转磷酸胆碱酶、1-磷酸半乳糖尿苷酸转移酶、钙蛋白酶、内酰胺酶等酶活性相关的基因下调,又揭示了血瘀体质者可能有生长发育下降的趋势。结论:血瘀体质理论体系对指导临床用药及养生保健具有重要意义。基因研究表明,与平和体质相比,血瘀体质者血小板易凝集,易患缺血性疾病和肿瘤类疾病;血瘀体质在生物合成与代谢功能、遗传信息传递方面的能力以及生长发育均有下降的趋势。更多还原

【Abstract】 Objective To excavate the related theories of Stagnant Blood Constitution(SBC) from Traditional Chinese Medicine theory,summarize contemporary research achievement and expatiate the theories of SBC and its application.To reveal the characteristics of gene expression for SBC by detecting the gene expression profiles of peripheral blood of SBC.It will provide theory basis to prevent related diseases early.Methods By the hods of the traditional literature research and the contemporary literature analysis,we discuss the correlative theories of SBC from origin to development,from connotation to extension.13 typical subjects of SBC and 30 subjects of normal constitution were selected on the basis of clinical epidemiological investigation.Ten milliliters of fasting venous blood were collected from each subject.The white blood cells were isolated,and the total RNA was extracted,purified and amplified.The complement RNA(cRNA) was labelled with biotin.The gene expression profiles of peripheral blood were detected with Sentrix Human-6 Expression BeadChip gene chip.The differentially expressed genes as compared with normal constitution were analyzed,and they were GO annotated and statistically pathway analyzed.The biological functions of differentially expressed genes were evaluated in order to reveal the cause,characteristics and liability of SBC at the level of gene.Results Theoretical studies found that the formation of blood stasis constitution was influenced by various of congenital and inherent factors,which included genetic factors, oldness,sex,tramatic injuries,seven emotions and internal injuries,improper diet, excessive labour and ease,and the prolonged disease.Genetic researches revealed that there were 592 differential gene locus expressed in blood stasis constitution group as compared with the balanced constitution group when P<0.05,which included 167 up-regulated gene locus and 425 down-regulated gene locus; When P<0.001,the differential gene locus expressed in blood stasis constitution group dcreased by 96 as compared with the balanced constitution group.Of these,24 were up-regulated gene locus and 72 were down-regulated gene locus.Patients with blood stasis constitution were more easily to have the platelet aggregation status,and had a high likelihood of suffering ischamic and tumor disease.A lot of biosynthesis-related genes which takes part in the process of protein,nucleic acid,and steroid hormone sythesis were down-regulated in population with blood stasis constitution,which indicates the decreasing status of genetic information transmission functions in blood stasis population.While the facts of many other genes which included growth and development-related and enzyme activity-related genes such as dihydropyrimidine dehydrogenase,human methyltransferase,protein-arginine n-methyltransferase,UDP glucose pyrophosphorylase,cholinephosphotransferase,phosphate galactose uridylic acid transferase,calpain,lactamase,down-regulated indicated again the possibility of decreasing in growth and development in population with blood stasis constitution.Conclusions The theoretical system of SBC could contribute to clinical medication and health preservation.There were significant differences of peripheral blood gene expression profile between the SBC and the normal constitution.The blood platelet of SBC is easier to aggregate and has the tendency to suffer from ischemic disease and cancer.The biosynthesis and metabolism function,the ability of genetic message transmission and the growth and development have downtrend.更多还原