| 【Purpose】 1.Through systematic research and analysis of literature on DNA methylation and TCM constitution and syndromes,clarify the relationship between epigenetics and TCM constitution,DNA methylation and kidney-yang deficiency constitution,and apply TCM to DNA methylation Physical fitness research provides theoretical support.2.Based on the spontaneous kidney-yang deficiency constitution model rats,methylated DNA immunoprecipitation technique was used to study the differential expression level of genome-wide DNA methylation of kidney-yang deficiency constitution model rats,and to explore the DNA methylation characteristics of kidneyyang deficiency constitution,so as to provide new tools and ideas for the further study of TCM constitution.【method】 1.Literature research(1)Literature search: "DNA methylation","physique","Chinese medicine","syndrome","syndrome","DNA methylation","constitution","physique","Chinese medicine"," "Syndrome" is the search term in Pub Med,Wanfang Data,Weipu,and China Zhinet to retrieve the documents published from the date of establishment of the database to February 28,2021.(2)Literature inclusion and exclusion: The literature is sorted according to the established literature inclusion and exclusion criteria.(3)Literature analysis: extract literature information,formulate tables,organize data,perform descriptive statistical analysis,and clarify the relationship between epigenetics and traditional Chinese medical constitution,DNA methylation and kidney-yang deficiency constitution.2.Experimental research(1)The first generation and the fourth generation of spontaneous kidney-yang deficiency constitution model of rats developed by our research group were selected to verify the model traits.(2)Three male rats were randomly selected from the normal group,the second generation and the fourth generation,and the liver tissues were collected for genomewide methylated DNA immunoprecipitation sequencing.(3)The DNA methylation sequencing results of the whole genome of rats with kidney Yang deficiency constitution model were compared with the reference genome,and the enrichment regions on the genome were located.The differences between groups were compared to find the differential methylation sites,and the gene associations of the differential methylation sites were conducted.(4)Primary screening of differential methylation sites and associated genes in kidney-yang deficiency model rats.【results】 1.Literature research(1)The research on DNA methylation and TCM constitution and syndromes has gradually become a new academic growth point in the field of TCM.Compared with the research on syndromes,the research on constitution and DNA methylation is obviously insufficient.(2)Epigenetics and TCM physique are consistent in theoretical basis and research context.(3)The formation of a new model of DNA methylation during pregnancy affects the formation of constitution,and DNA methylation is closely related to the constitution of kidney-yang deficiency.2.Experimental research(1)Evaluation of the character of kidney yang deficiency in animal model of kidney yang deficiency,and the model was established successfully Macro behavioral evaluation: the rats of the first and fourth generations were in a good state of mind,with darker eyes,dull reaction,dark and messy coat color,reduced activity,huddling with bow and back,and perianal pollution.(2)Genome-wide DNA methylation sequencing of liver tissues The DNA samples were tested,and the results showed that the samples basically met the sequencing conditions.The original data obtained by sequencing were filtered,and the quality control analysis and evaluation of the processed data were carried out.The results showed that the quality of the data was qualified and the conditions of data analysis were met.A total of 860,000 differential methylation sites were found by bioinformatics analysis,and the data analysis results were reliable.(3)Bioinformatics analysis of genome-wide DNA methylation differential sites in kidney-yang deficiency rats(1)Genome enrichment region mapping results: a total of 276091 peak,samples were obtained in the offspring of kidney-yang deficiency rats,309430 peak,and 103143 peak,were obtained in the fourth generation of kidney-yang deficiency rats,and 275413 peak,were obtained in the normal group.An average of 91804 peak,samples were obtained.There was no significant difference in the overall enrichment degree of genomes among the three groups.(2)The results of differential peak analysis: there were 372 peak differences between the first generation kidney-yang deficiency rats and the normal rats,of which 89 were of methylation level increased and 283 of which were decreased,there were 51 peak differences between the fourth-generation kidney-yang deficiency rats and the normal rats,of which 30 were methylation level increased and 21 were methylation level decreased.The number of differential methylation sites in the first generation was significantly higher than that in the fourth generation.(3)The results of peak analysis of differences between groups: the difference peak of the first generation of kidney-yang deficiency rats was compared with that of the fourth generation of kidney-yang deficiency rats,and 12 identical differential peak,were obtained and associated to 11 genes.The increased methylation levels of Gdap1,Cdkal1 and Cd300lb;were Zmiz1,Fam111 a,Sh3bp4,As3 mt,Col16a1,LOC103690271,Wdpcp,LOC102553861.Functional annotation and classification of related genes were carried out in Go and KEGG databases,of which 10 were included in Go database(not included in LOC102553861 and not included in),KEGG database at present.(4)Twelve sites with different DNA methylation differences in kidney Yang deficiency constitution were preliminarily screened,and 11 genes were closely related.【conclusion】 1.The formation of a new model of DNA methylation during pregnancy affects the formation of constitution,and DNA methylation is closely related to the constitution of kidney-yang deficiency.2.The differential expression of DNA methylation exists in kidney Yang deficiency constitution model rats,which partly explains the formation and development of TCM constitution and the scientific nature of TCM theory from the perspective of epigenetic modification,and provides new ideas and tools for the in-depth study of TCM constitution.3.From the degree of differential expression of DNA methylation,it is preliminarily proved that the first-generation model rats have more prominent characteristics of kidney yang deficiency than the fourth generation model rats,and the idea of offspring modeling is more in line with the needs of TCM constitution modeling.4.The primary screening of kidney-yang deficiency model rats showed 12 differential methylation sites,which were closely related to 11 genes,whose functions were concentrated in mitochondrial energy metabolism,glycolipid metabolism and tumorigenesis.Among them,the newly discovered 4 genes have not been reported in relevant functional studies at home and abroad.This study preliminarily confirmed that these genes may be related to kidney Yang deficiency constitution,providing a new perspective for the subsequent basic research on constitution biology of traditional Chinese medicine. |
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