| 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. |
PDF Full Text Request