【作者】 张燕；

【导师】 罗炳德；

【作者基本信息】 南方医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 研究背景20世纪以来,随着人类的平均预期寿命的不断延长,世界人口结构的老龄化趋势日益明显,一个人口老龄化的时代已经来临,专家预测人口老龄化的高峰将出现在21世纪中期,届时老年人庞大的医疗费用支出将成为各国的沉重负担,而解决这个问题的唯一途径就是提高老年人的健康水平。这清楚表明关于衰老和抗衰老研究在21世纪国际医学研究中的重大意义,事实上近二十年来该课题的研究已成为世界各国医学研究的热点前沿之一,因此寻找理想的抗衰老措施使老年人延缓衰老,保持健康具有重大社会意义。我国传统中医药对衰老有独特的认识,针灸抗衰老是中医的优势所在。古今的临床及近现代实验研究都表明灸法具有良好的抗衰老作用,灸法作为一种传统疗法,具有延缓衰老的作用,这已被大量文献所证实,命门穴位是最常用的抗衰老穴位之一。1953年DNA双螺旋结构的发现以及1985年Crick的遗传信息传递的中心法则的提出后,生命科学获得了极大发展,基因芯片技术是全基因组分析的有力技术平台,是一种对基因序列及功能进行大规模、高通量的研究方法,能够并行分析大量基因,全面了解各个基因的表达变化,从而迅速获得丰富、全面的生物学信息,由此促进了基因组学的发展,如何充分利用现代科学技术的方法和手段,将传统针灸的优势与现代先进的科学研究技术有效地结合,从基因表达水平的物质基础研究方面阐明针灸的作用机理,实现针灸现代化,已成为针灸研究领域的主要发展方向之一。针灸是在中医的脏腑经脉等相关理论的指导下,以针刺或艾灸刺激体表的特定部位（腧穴）,调动机体本身固有的调节功能,从而对机体的生理、病理过程产生影响以及这种影响在体内产生反应,达到治病的目的,对针灸刺激在机体产生的生物反应启动、过程、结果进行的研究称之为针灸作用原理研究,以寻求针灸作用的规律及其代表的生物过程的响应规律与机制,目前针灸学作为生命科学的重要组成部分,在其针灸作用原理现代化研究进程中,充分吸收、借鉴了生命科学领域的新思维、新理论、新技术,在针刺镇痛机理研究、经络和腧穴本质探讨方面取得了一系列进展,针刺与神经内分泌、免疫系统相关性研究成果也已为医学界所认同,在针刺研究方面我们看到了可喜的成果。作为同样古老有效的治疗方法,目前灸法抗衰老的现代研究大部分是在细胞水平进行研究,在相关调控基因表达的研究局限于选取数个基因进行研究,其研究深度远远不够,不能较全面地反应这一前提下灸法治疗后基因表达的全貌,在现代分子生物学的迅速发展下,这种研究方式有一定的局限性,不能体现灸法理论现代化的发展,基因芯片一次能检测上万条基因,为灸法抗衰老在基因水平进行全面的机理研究提供了技术支持。目的衰老的机制是极其复杂的,可能涉及的基因较多,而灸法是通过穴位对机体进行整体调节,其机制亦可能涉及较多基因,灸法治疗在全基因组水平影响哪些靶基因,尚无人研究,本研究拟采用大鼠全基因组芯片对老年大鼠和年轻大鼠的血液白细胞基因表达谱进行分析,目的在于尽可能全面的了解导致老年状态的基因背景,同时我们进一步分析了灸命门穴位后老年大鼠血液白细胞基因表达谱的改变,了解灸命门穴位延缓衰老的基因调控模式,全面的阐明灸命门穴位延缓衰老的分子作用机制,为灸法延缓衰老在全基因组学方面提供新的理论依据,从基因组整体水平上揭示艾灸命门穴位延缓衰老的物质基础,在灸疗作用原理研究领域寻找新突破口。方法1文献研究,采用评述的方法,整理灸法延缓衰老的研究现状,分析其机制研究的不足,以期理清本实验的研究思路。2实验研究,采用南方医科大学实验动物中心提供的SPF级雄性Wistar大鼠30只,其中18月龄老年大鼠20只,随机分为2组（每组各10只）,即老年对照组（老年组）、灸命门穴组（灸疗组）;另设4月龄Wistar大鼠10只作为年轻对照组,设为年轻组。灸疗组每天温和灸命门穴1次,每次灸8min,老年组、年轻组不予处理。灸疗组持续治疗10d,治疗结束后,各组大鼠以水合氯醛腹腔麻醉后,心脏取血,血液抗凝后从血液中提取白细胞,然后从白细胞中提取总RNA,对提取的总RNA质量鉴定合格后,进入芯片检测,先反转录合成第一链cDNA和第二链cDNA,cDNA纯化后接着体外转录合成cRNA,然后进行cRNA纯化,之后进行杂交,洗涤,染色,具体过程遵守Illumina公司基因表达谱操作程序。所用芯片是大鼠全基因组芯片,一共22106个探针。应用illumina微珠芯片扫描仪扫描芯片信号,应用illumina微珠芯片分析应用软件BeadStudio对芯片灰度扫描图进行分析,将图片的点阵转化为数字型数据,分别得到老年组与年轻组比较以及灸疗组与年轻组比较的血液白细胞基因表达谱,按照GeneOntology分类标准,对老年组与年轻组比较的差异基因进行生物信息学的基因功能显著性分析,从而了解造成老年大鼠与年轻大鼠生物学差异的靶向性基因功能及其所属基因,并且对这些所属差异基因在灸命门穴后表达的变化进行分析,另外随机选择差异基因进行荧光定量实验验证基因芯片,选择基因CCL4、SOD2、ADORA2A作为目的基因,选择GAPDH作为管家基因,引物序列设计软件为ABI primer express 3.0,由上海英骏生物技术有限公司进行引物序列合成后,采用ABIPRISM7300序列检测仪,进行荧光定量RT-PCR检测,观察溶解曲线和扩增曲线,使用相对定量△△Ct法进行数据分析,验证芯片结果的准确性。结果1文献研究自黄帝内经开始就有关于艾灸益寿延年及命门穴可补肾培元延缓衰老的有关记载,千百年来灸法治疗在中华名族的身体健康方面作出了巨大的贡献。在中医现代化的发展中,就目前灸法延缓衰老的机理研究现状而言,大多集中在细胞水平方面,在相关调控基因表达的研究局限于选取数个基因进行研究,还没有开展全基因组水平的研究,因此以大鼠的全基因组基因芯片研究灸命门穴后大鼠血液白细胞基因表达谱的变化,可为灸法延缓衰老在基因组学方面提供新的理论依据,在灸疗作用原理研究领域寻找新突破口。2实验研究（1）灸命门穴在全基因组水平延缓衰老血液白细胞基因谱的比较中,老年组与年轻组比较有436条基因表达显著差异,灸疗组与年轻组比较有76条基因表达显著差异,说明灸疗组、年轻组、老年组在血液白细胞中有彼此区别的基因表达特征,灸命门穴位使老年组异常基因的表达水平趋向年轻组,灸疗组在整体基因表达上趋同于年轻组,提示灸命门穴使老年鼠的基因表达谱移向年轻鼠基因表达谱,从全基因组水平说明灸命门穴位可以延缓衰老。（2）大鼠血液白细胞衰老的靶向性基因功能及相关基因老年大鼠与年轻大鼠的血液白细胞基因谱比较,有436条基因表达差异,其中表达上调的基因有289条,表达下调的基因有147条。运用生物信息学分析方法对这组差异基因进行了基因功能显著性分析,结果表明造成老年大鼠与年轻大鼠生物学差异的靶向性基因功能有:①氧和活性氧中间物参与新陈代谢过程基因功能,其中含有9条基因,表达显著下调,例如SOD2、TXNIP、GPX3、PPIF等;②炎性反应基因功能,其中含有22条基因,表达显著上调,例如LTA、TGFB1、ZBTB7A、SH2D2A、CCL5等;③凋亡基因功能,其中含有28条基因,表达显著上调,例如F2R、BIRC3、DDIT3、ADORA2A、HERPUD1等。（3）灸命门穴对老年大鼠血液白细胞基因表达的影响灸命门穴干预后的灸疗组与年轻组的血液白细胞基因谱比较,有76条基因表达差异,其中表达上调基因有65条,表达下调基因有11条。灸命门穴位治疗后,我们发现在老年组与年轻组比较中属于显著靶向性基因功能的差异表达基因大部分在灸疗组与年轻组的比较中正常表达或者趋向正常表达,灸命门穴位调控老年大鼠血液白细胞基因表达的作用模式有以下几个特点:①灸命门穴位上调老年组中涉及氧和活性氧中间物参与新陈代谢过程基因,其中8条基因在灸疗组正常表达,例如SOD2、TXNIP、GPX3、PPIF等基因。②灸命门穴位下调老年组参与炎性反应的基因,其中5条在灸疗组中趋向正常表达,例如CCL5、EGR1、FCGR3A等基因,14条炎性反应基因在灸疗组中正常表达,例如LTA、TGFB1、ZBTB7A、SH2D2A等基因。③灸命门穴位下调老年组中凋亡基因的表达,其中3条基因例如PRF1、GZMB、ADORA2A等在灸疗组中趋向正常表达,24条基因在灸疗组中正常表达,例如CEBPB、BIRC3、GADD45B等基因。（3）荧光定量RT-PCR实验各基因扩增曲线平滑,每条曲线均有明显的指数扩增期,说明PCR检测具有较好的敏感性。各基因溶解曲线均为单一峰,未出现其它异常波形,说明PCR扩增特异性较好。使用相对定量△△Ct法进行数据分析各基因原始模板浓度2^-△Ct,表明基因表达的变化与芯片结果一致,芯片结果可靠。结论本研究首次利用现代科学全基因组基因芯片技术研究灸命门穴后大鼠血液白细胞基因表达谱的变化,在灸疗作用原理研究领域为灸法延缓衰老在全基因组学方面提供新的理论依据,研究结论如下:1本研究在全基因组学水平证明艾灸命门穴位可以延缓衰老。2本研究在全基因组学水揭示艾灸命门穴位延缓衰老的相关反应性基因。3本研究在全基因组学水平揭示艾灸命门穴上调抗氧化基因抗衰老,反应性基因是SOD2、GPX3、ATOX1等8条基因。4本研究在全基因组学水平揭示灸命门穴下调凋亡基因抗衰老,反应性基因是FOXO1A、RUNX3、F2R、ADORA2A等27条基因。5本研究在全基因组学水平揭示灸命门穴下调炎性反应基因抗衰老,反应性基因是CCL5、ICAM1、TAP2等19条基因。总之,本研究将传统灸法抗衰老的优势与现代先进的全基因组基因芯片技术有效地结合,在全基因组范围内发现艾灸治疗的反应性靶基因,从基因表达水平的物质基础研究方面阐明灸疗的作用机理,揭示灸命门穴延缓衰老的作用模式是一种多基因多靶点的整体调整作用的特点,在灸疗机理现代化研究的路上迈上了重要的一步,希望抛砖引玉,为将来灸疗作用机理的实验研究提供新的思路和方法学参考,灸法延缓衰老的分子生物学研究,对抗衰老的靶基因及其临床应用也有积极意义。更多还原

【Abstract】 Backgroundfrom the 20th century, With the average life expectancy of human continuous extension, The structure of the world’s population aging trend is increasingly clear,and comes An aging population era. experts predict that the population of the elderly population will reach a new height in the the mid-21st century, By then the elderly large amount of medical expenses will become a heavy burden, and the only way that would get rid of this problem is to raise the level of health tolerance of our elderly. The research conducted by the 21st Century International Medical Assossication clearly shows the direct relationship of aging and antiaging. As a matter of fact, in the past 20 years this research has become a hot topic within the medical science community, we should find a ideal anti-aging ways that would benefit much as a society. Traditional Chinese medicine in our country has a unique understanding of aging, acupuncture always have the advantages in antiaging in TCM, Old and modern clinical and experimental studies show that moxibustion has a good anti-aging effect, Moxibustion is a traditional therapy with the role of anti-aging, it has been confirmed by a large number of documents, Mingmen point is the most commonly used acupuncture points on anti-aging. In 1953 DNA double helix structure had been discovered and in 1985 Crick told the genetic information transmitted to the central dogma, the life sciences have been a great development, genechip technology is the whole-genome analysis of a powerful technology platform, is a gene sequence and function of large-scale, high-throughput research methods, be able to parallel analysis of a large number of genes, a comprehensive understanding of the various changes in gene expression, thus the rapid access to rich comprehensive biological information, promoting the development of genomics, How to take full advantage of modern scientific and technological ways and means, integrate the advantages of traditional acupuncture with modern advanced technology to go on effective scientific research,at the level of gene expression from the basic material research to clarify the mechanism of acupuncture,acupuncture modernization has become one of the main direction of Acupuncture Research, under the guidance of the the zangfu meridians theory, Acupuncture is to stimulate specific parts of the body surface （acupoints） with acupuncture or moxibustion, to mobilize inherent in the regulation of body functions, thus lead to the body’s physiological and pathological processes, as well as have an impact on this species and have affected the response in the body to achieve the purpose of medical treatment, use Acupuncture stimulation at the body to get the biological response ,how to start the process, what are the results of the study,what is the role of the principle in the acupuncture study, to find the role of the law and their representatives how to respond to the laws of biological processes and mechanisms. At present, as the an important part of life sciences ,acupuncture use the full absorption of the life sciences draw on new ideas, new theories and new technologies on the study about the role of the principle of acupuncture in its modernization process, in the study of Mechanism of acupuncture analgesia, the nature of meridians and acupoints, a series of progresses have been made, the results about the relations of acupuncture and neuro-endocrine or immune system have also been recognized by the medical field, we have good results in acupuncture research. As the same old methods of effective treatment, the most modern research on moxibustion Anti-Aging is currently to study at cellular level, study of the relevant gene expression limited to a few select genes, the depth of their research is far from enough,Can not respond to a more comprehensive manner of the gene expression after moxibustion treatment .with the rapid development of modern molecular biology , this kind of the research has some limitations, should not reflect the modern theory of the development of moxibustion, Gene chip can detect genes over a 10,000 genes at a time, has provided technical support for a comprehensive study the anti-aging mechanism of moxibustion at gene level.ObjectiveThe aging mechanism is extremely complicated and may involve more genes, And moxibustion through the points on the body to conduct an overall adjustment,its mechanism may involve more genes too, there have not been study on how many target genes affected by the Moxibustion at the genome-wide level, In this study, the rat whole-genome chip was used to analysis the blood leukocyte gene expression profiles in the old rats compared to the young rats , The aim is to understand the genetic background in the old rats as complete as possible , At the same time, we further analysis the changes in blood leukocyte gene expression profiles after Moxibustion on Mingmen point in old rats , understanding the gene regulation model of anti-aging by Moxibustion on Mingmen points, set out a comprehensive molecular mechanism of the anti-aging effection with Moxibustion on mingmen point, provide a new theoretical basis of Anti-aging molecular mechanism of moxibustion at genome-wide study, From the genome level to reveal the material basis for anti-aging with moxibustion on Mingmen point, find a new breakthrough in moxibustion research areas.Methods1 Literature Researchwe used the Review of the methods and organize the anti-aging research on Moxibustion, Analysis of the lack of research into the mechanism of aging of modern research, in order to clarify the research ideas in this experiment.2 Experimental StudyThirty SPF male Wistar rats were supplied by the Experimental Animal Center of Southern Medical University, among whom, 20 rats were 18 months old divided into a old control group and a old moxibustion group with 10 in each group. The other 10 rats were 4 months old as a young control group . The old moxibustion group was treated by the mild warm moxibustion on the Mingmen points with one treatment lasting 8 minutes and one treatment a day for total 10 days. The old control group and the young control group were not treated. After the old moxibustion group completed the treatment, all rats were anesthetized with intra-peritoneal chloral hydrate and blood samples were collected via cardiac puncture and anticoagulated ,then the leucocyte and the total RNA were extracted for gene expression profile analysis, after The total RNA quality accreditation. Then into the chip testing, After reverse transcription from RNA to first-strand cDNA and then second-strand cDNA, the following procedures were performed according to the Illumina gene expression analysis operating manual: cDNA purification, in vitro transcription to cRNA, cRNA purification and hybridization, washing, staining and scanning, the Expression BeadChip, a rat whole genome chip that contains a total of 22106 probes, was used in the study. The arrays were scanned using an Illumina BeadStation scanner and the data were extracted using Illumina BeadStudio software. Pictures of the lattice will be converted into digital data, get the gene expression profiles of the blood leukocyte in Old group compared to the young group and in the moxibustion group compared to the young group respectively, According to the Gene Ontology Classifications, we carry through the gene function differential analysis of bioinformation on the gene expressions in the old group compared to the young group , in order to understand the targeted gene function and their genes which lead to the biological differences between young rats and the old rats,and analyse the gene expression change of the old rats after the moxibustion on mingmen point, we randomly selected differential genes for quantitative fluorescence RT-PCR to verify the gene chip. Select gene CCL4, SOD2, ADORA2A as target genes and GAPDH as a housekeeper gene. The ABI primer express 3.0 software were used to design the primer sequences and the primer sequences were synthesized by Shanghai Invitrogen Biotechnology Co. Ltd, SYBR Green RT-PCR was performed using ABI Prism 7300. dissociation curves and amplification curves were Observed.the relative quantitativeΔΔCt method was used to analyze the results.Results1 Literature Research since Huang Di Nei Jing era,there have been many anti-aging records about the antiaging effect of moxibustion and Mingmen point, Moxibustion treatment has made enormous contributions to the Chinese in good health for thousands of years. In the development of modernization of traditional Chinese medicine,the current research on the anti-aging mechanism of moxibustion mostly concentrated in the cellular level, in the regulation of gene expression related to the study limited to a few select genes, has not been the level of whole-genome, So we use the rat genome-wide gene chips in the study, to know changes in gene expression profiles of rats blood leukocyte after Moxibustion on Mingmen point,We provided a new theoretical basis for the antioxidation and anti-aging effects of moxibustion at a genomic level and represents a breakthrough in the research on the mechanism of moxibustion.2. Experimental Study（1）Moxibustion Mingmen points antiaging at the level of genome-wide level.Compared to the juvenile control group, 76 differential genes were identified in the senile moxibustion group, but 436 in the senile control group. In other words, if one considers the juvenile control group as normal, the treatment of moxibustion on the Mingmen point in the senile group recovered the abnormal gene expressions to normal. Moxibustion on Mingmen point enable gene expression levels of the old group tend to the younger group, the gene expressions of the moxibustion group in the overall convergence in the younger group,It prove that moxibustion on the Mingmen point has an anti-aging effect at the genome-wide level.（2） target gene functions and genes Cause biological differences between the aged rats and young ratsThere were 436 genes with different expressions between the senile control group and the juvenile control group, There are 289 genes increased expressions and 147 genes reduced expressions. the significant differential gene function analysis of the Bioinformatics: the character of the gene function expression mode in the old rats leukocyte compared to the gene expression of the young rats leukocyte, target gene function as follows:①.9 genes was differentially down -regulated expressions with the function that are involved in oxygen and reactive oxygen intermediates metabolism,such as SOD2、TXNIP、GPX3、PPIF and so on. .②22 genes was differentially down -regulated expressions with the function that are involved in inflammatory response,such as LTA、TGFB1、ZBTB7A、SH2D2A、CCL5 and so on.③28 Apoptosis genes was differentially downregulated expressions, such as F2R、BIRC3、DDIT3、ADORA2A、HERPUD1 and so on.（3） the changes of blood leukocyte gene expressions after Moxibustion on the mingmen point of aged rats there are 76 differential genes in the old moxibustion group compared to the young control group, There are 65 genes increased expressions and 11 genes reduced expressions. After moxibustion on mingmen point, we found that most of the differential genes with remarkable function in the old group have expressed normal or closer to normal in the moxibustion group, the Several characters of the leukocyte gene expression changes of the rats are as follows:①moxibustion on mingmen point can differentially up-regulate the genes involved in oxygen and reactive oxygen intermediates metabolism in the old group compared to the young group, eight of the genes restored to normal in the moxibustion group,such as SOD2、TXNIP、GPX3、PPIF and so on.②moxibustion on mingmen point can differentially down-regulate the genes involved in inflammatory response in the old group compared to the young group, five genes are closer to normal expression in the moxibustion group, such asCCL5、EGR1、FCGR3A and 14 genes expressed normally in the moxibustion group,such as LTA、TGFB1、ZBTB7A、SH2D2A and so on.③Moxibustion on mingmen point can differentially down-regulate the genes involved in apoptosis in the old group compared to the young group, 24 genes showed normal expressions in the old moxibustion group such as CEBPB、BIRC3、GADD45B and so on, 3 genes are closer to normal expression in the moxibustion group such as PRF1、GZMB、ADORA2A..（3） The results of the RT-PCR verification:the gene amplification curves are smooth, and each curve of the index were significantly amplified by a view that PCR has better sensitivity of detection, the genes Dissociation curves are single-peak curve, no other abnormal waveforms, a better description of specific PCR amplification,the relative quantitativeΔΔCt method was used to analyze the original template concentration 2^-ΔCt. The results were consistent with that of gene chip study, indicate the results of genechip are reliable.Conclusionthis gene-chip technology was used to study the changes of blood leukocyte gene expression profiles in rats after the Moxibustion on Mingmen point,provides new theoretical basis for anti-aging at the whole genome level, the conclusions are as follows :1. Firstly the whole-genome gene-chip technology was used for the mechanism of anti-aging research of moxibustion.2. Firstly at the level of genome-wide study revealed the aging delay effect of moxibustion on Mingmen point.3 Firstly at the level of genome-wide study revealed the target anti-aging genes of moxibustion on Mingmen point.4 Firstly at the level of genome-wide study revealed moxibustion on mingmen point upregulated the genes related to oxidation for antiaging,target genes including eight genes of SOD2、GPX3、ATOX1 and so on.5 Firstly at the level of genome-wide study revealed moxibustion on mingmen point downregulated the genes related to apoptasis for antiaging, target genes including 27 genes of FOXO1A、RUNX3、F2R、ADORA2A and so on.6 Firstly at the level of genome-wide study revealed moxibustion on mingmen point downregulated the genes related to inflammatory response for antiaging, target genes including 19 genes of and CCL5、ICAM1、TAP2 and so on. In short, this study effectively integrated the traditional anti-aging advantages of moxibustion with modern genome-wide gene-chip technology, at the whole genome-wide level find target genes response to treatment, at the gene expression levels from basic materials to clarify the mechanism of moxibustion, revealed the antiaging of Moxibustion on Mingmen point reflected a pattern of many target genes and many of the characteristics of the role of overall adjustment, go a an important step on the road to the modern research of acupuncture, provided a new ideas and methods of study reference for the future study of the mechanism of moxibustion Experimental study.更多还原