| Antler is the only bone organ that can regenerate completely in mammals.In its rapid growth period,the growth rate of antler antler antler can reach 2 cm per day,and the cell proliferation rate is more than 30 times that of cancer cells,which can be said to be incomparable with any tissue and organ in mammals.However,there is no sign of canceration in antler cells,so understanding the function and role of growth factors in the process of antler growth can provide new ideas and directions for human medical research(such as tumor treatment).At present,it has become the focus of scientific research to explore and clarify the internal molecular mechanism of velvet antler proliferation,ossification and apoptosis.Antler apical tissue is the center of antler growth and differentiation,which can be divided into five tissue layers:antler cortex,mesenchymal layer,anterior cartilage layer,transition layer and cartilage layer.The research on the role of each tissue layer in the process of antler growth is mostly focused on the mesenchymal layer,while the research on other layers is relatively less.The occurrence and regeneration of pilose antler skin has a unique mechanism,and has a strong auxiliary effect on the occurrence and regeneration of pilose antler.Sika deer,as a typical representative of the deer family,provides a good research material for the growth and development of deer antler.As the main form of epigenetic modification,DNA methylation can regulate the transcription and expression of target genes,and then play a regulatory role in the process of life.Therefore,it is also an important scientific field to study the promoter methylation of the related genes in the skin of sika deer antler from the dynamic point of view of the growth process of sika deer antler.In this study,sika deer were selected as the research object,and the top antler skin tissues of three typical dynamic time points in the process of antler growth were used as experimental materials to carry out the gene promoter of insulin-like growth factor(IGF 1),nerve growth factor(NGF)and keratinocyte growth factor(KGF)related to the regulation of antler growth On this basis,we used BSP technology to study the DNA methylation patterns of the three genes in Sika deer antler top skin tissue,and then analyzed the differences of DNA methylation in the three tissues and explored the regulatory mechanism:1.The partial promoter sequences of IGF1,NGF and KGF genes from sika deer were cloned,and all of them had typical characteristics of core promoter element,CpG island region and core promoter region or site.2.IGF1,NGF and KGF genes showed different DNA methylation patterns in the antler skin during the growth of antler,and the methylation rate ranged from(0.00 ± 0.00)%to(48.88±4.44)%.The methylation rates of NGF gene were(23.99±0.66)%,(22.22±1.78)%,(15.10 ±1.55)%,respectively.The methylation rates of IGF 1 and KGF gene were(15.55 ±1.11)%,(48.88 ±4.44)%in the expression stage,respectively Methylation in the middle and late stage.3.The methylation patterns of IGF1,NGF and KGF genes,as well as the DNA methylation patterns of the same gene promoter region in different deer skin tissues,are affected by the methylation degree of CpG dinucleotide sites in their respective promoter sequences.4.IGF1 and KGF genes were methylated in different degrees in the antler skin of sika deer in the expression stage,and were highly expressed in the middle and late stage.There was no methylation,The methylation rate of the two genes in the expression stage was significantly higher than that in the middle and late stage(P<0.05).There were 12 CG sites in the promoter region of IGF1 gene.At 27bp and 82bp,there was only expression stage hypermethylation,which methylation rate was significantly higher than middle and late stage((P<0.05).There were three CG sites in the promoter region of KGF gene.The methylation of 169bp,242bp and 354bp occurred in antler skin tissue in the expression stage,but no methylation occurred in the middle and late stage,The methylation rate in expression stage was significantly higher than that in middle and late stage(P<0.05).5.NGF gene was methylated in different degrees in antler skin tissoes,but there was no significant difference between them(P>0.05).There were 30 CG sites in the promoter region of NGF gene,which were methylated at 35bp,39bp,42bp,52bp,66bp and 95bp,but the difference was not significant(P>0.05).The methylation rate at 115bp,125bp and 135bp in early stage was significantly higher than that in late stage(P<0.05),and there was no significant difference in the early and middle,middle and late stages(P>0.05).The methylation rates at 223bp,225bp and 227bp were significantly higher than those at the late stage(P<0.05),but there was no significant difference between the two stages(P>0.05).At 246bp,there were significant differences in the methylation rate between the expression stage and the middle stage,the methylation rate between the middle stage and the late stage,and the methylation rate between the expression stage and the late stage(P<0.01).The methlation rate at the expression stage of 347bp was significantly higher than that at the middle and late stages(P<0.05),but there was no significant difference in the methylation rate between middle and late stages(P>0.05).There was no significant difference in the methylation rate of other CG sites(P>0.05).In this study,from the dynamic point of view of the growth process of sika deer antler,the top antler skin tissue was used as the experimental material,and the results of DNA methylation research on the genes related to the growth regulation of sika deer antler provided the basic theoretical accumulation for revealing the regulation mechanism of the growth and development process of sika deer antler,and also for studying the regulation mechanism of proliferation,ossification,apoptosis and tissue and organ regeneration of sika deer antler,restoration,etc. |
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