| Research background:Radiation immunology mainly explores the effects of ionizing radiation on the immune organs,immune cells and immune molecules in the body.Under normal circumstances,the body's immune system is in an auto-stabilized state.When exposed to radiation,due to different doses,it can cause significant changes in the surface molecules,cytokines,and signaling molecules of immune cells.These changes are the important basis of radiation immune effect of ionization.High dose radiation will inhibit the proliferation of thymocytes and lymphocytes,and the related surface molecules and cytokines will also change.Eventually,it will do harm to the body's immune system.On the contrary,low-dose radiation stimulates the maturation and proliferation of thymocytes and lymphocytes,and stimulates the secretion of lymphocyte factors,which will induce the excitatory effect of the immune system.Functional genomics is a new stage in genomics research.It is based on the rich information resources provided by structural genomics.It uses advanced gene expression technology,biological function detection technology and bioinformatics technology to study gene expression,regulation and function.In the field of genomics development,there are many basic research fields in immunology,especially the network pattern of cytokine interaction in different immune response states,the related cell signaling network mechanism in immune cell development and activation,and the importance of immune response.Research on plasticity and other aspects can not be separated from the support of high-throughput and large-scale genomics.Current research has found that epigenetic modifications are also involved in the regulation of immune responses.Epigenetic modification refers to the heritable changes in gene expression without changes in the nucleotide sequence,including DNA methylation,histone modification,and RNA editing.Epigenetic modifications of cells undergo dynamic changes during their maturation and function developmentto ensure that gene expression is precisely regulated.Therefore,an in-depth study of epigenetic regulation mechanisms of immune responses is helpful to elucidate the molecular mechanisms underlying radiation immune effects,and fills in new theoretical basis for radiation immunology theory.Research objectives:1.To investigate the differential expression and functional analysis of Th1-Th2-Th3 type cell related genes in mouse thymus induced by different doses of ionizing radiation,and to elucidate the molecular mechanisms of high and low dose radiation on T lymphocyte differentiation and immune response;2.Screening differentially expressed genes in mouse spleen whole gene expression profiles induced by different doses of ionizing radiation,and performing bioinformatics analysis to verify immune response-related cytokines,co-stimulatory molecules and TGF-? signaling pathway-related genes,and further Explore the regulatory mechanisms of TGF-? signaling pathways;3.To investigate the effects of epigenetic modifications related genes on immune cells in mice irradiated with different doses of ionizing radiation,and to screen the key Lnc RNAs in mouse thymus Lnc RNA and m RNA expression profiles at different doses of ionizing radiation,and perform bioinformatics analysis.The Mechanism of Lnc RNA and Target Gene Coexpression Networks in Radiation Effects.Research methods:1.By using the PCR array chip technology to detected the differential expression of Th1-Th2-Th3 type cell related genes in mouse thymocytes of 24 h after high-and low-dose X-ray irradiation.ELISA and immunofluorescence co-localization methods were used to detect secretion of IFN-?,IL-10,TGF-?,IL-17 a and IL-21,different cytokines of different types of Th cells at different doses and at different time points after irradiation.Surface antigen expression was detected;2.High-throughput whole-gene expression microarray technology was used to screen differentially expressed genes in mouse splenocytes 24 hours after whole-body irradiation with high and low doses of X-rays,differentially expressed genes,related pathways,and gene functions(Gene Ontology,GO)performs bioinformatics analysis to predict function of differentially expressed genes.Then,the effect of different dosesof ionizing radiation on the expression of cytokines and co-stimulatory molecules in mouse spleen was detected by ELISA.Finally,the q RT-PCR method was used to detect the time course changes of TGF-? signaling pathway related genes in Mouse spleen cells,thymocytes and in vitro EL-4 and Jurkat cells after high and low doses of X-rays,and DMRIE-C(transfection reagent specific for suspension cells)was used.Transient transfection method,si USP15 fragment was transfected into Jurkat cells,after high and low doses of X-rays,to explore the role of USP15 on TGF? signal transduction pathway;3.The q RT-PCR method was used to verify the genes associated with epigenetic modifications in mouse spleen cells,thymocytes and in vitro EL-4 cells treated with high and low doses of X-rays.Then,Lnc Path TM gene chip technology was used to screen the key Lnc RNA-m RNA co-expressed genes in mouse thymocytes 24 h after total doses of high-and low-dose X-rays,and differentially expressed Lnc RNA and related signaling pathways(Pathway)and Gene Ontology(GO)were used for bioinformatics analysis to predict the function of differentially expressed Lnc RNA-m RNA co-expressed genes and then validated using q RT-PCR.On this basis,the time course changes of EZH2 and the expression of downstream signaling molecules were investigated by q RT-PCR and Western blot.Research Results:1.Functional analysis of Th1-Th2-Th3 related genes in mouse thymocytes induced by different doses of radiation(1)In the LDR group(0.075 Gy),8 genes were up-regulated and 5 genes were down-regulated in mouse thymocytes;compared with the control group,54 were observed in the HDR group(2.0 Gy).Up-regulation of gene expression and down-regulation of 3 genes.Among them,LDR mainly induce the up-regulation of STAT4 genes,which play an important role in Th1 cells,and downregulate the GATA3,an important transcription factor of Th2 cells,and the TGF-?,a flagging cytokine of Th3/Tr1 cells.T cells differentiate into Th1 cells and induce a Th1-type immune response.After HDR,the up-regulated genes include positively regulated genes(IL-12 b,IL-15,IL-18,Gm-CSF,etc.)and negatively regulated genes(IL-5,TGF-?,IL-10,IL-6,IL-17,IL-23,etc.)simultaneously upregulate the Th2-type immuneresponse genes Cd86 and Irf4,so that T cells differentiate into Th3/Tr1 cells and induce a Th2-type immune response.(2)The IFN-?,IL-10,TGF-?,IL-17 a,IL-21 protein levels of different cytokines secreted by different Th cells in mouse thymocytes were detected by ELISA.At the same time,immunofluorescence co-localization method was used to detect the surface antigen of regulatory T cells(Th3/Tr1).After LDR,the expression of IFN-? secreted by Th1 cells was significantly increased,while other subtypes of Th cell-associated factors,except for IL-10,were decreased in different degrees.And the surface markers of regulatory T cells-decreased expression of CD25 and NRP1.However,after exposure to HDR,the secretion of IL-10,TGF-?,IL-17 a and IL-21 increased significantly,while the secretion of IFN-? secreted by Th1 cells decreased significantly.And the expression of cell surface antigens CD25 and NRP1 increased.It can be seen that different doses of ionizing radiation have different effects on the secretion of representative cytokines of various subtype Th cells in the thymus of mice.2.Whole gene expression profile of spleen in mice induced by different doses of radiation(1)Whole gene expression profiling analysis revealed that 1037 differentially expressed genes,of which 542 were up-regulated and 495 were down-regulated,were compared with the control group after mouse spleen cells were exposed to LDR.After exposure to HDR,compared with the control group,4831 genes were differentially expressed in mouse spleen cells,of which 2357 were up-regulated and 2474 were down-regulated.Of the 542 up-regulated genes induced by low-dose X-rays,12 genes overlapped with down-regulated genes induced by HDR;of the 495 down-regulated genes,there were 3 genes and high doses.X-ray-induced up-regulation of gene overlap includes genes involved in biological processes such as DNA methylation,ubiquitination,protein-DNA complex recombination,DNA modification,and cellular immunity.Further analysis of KEGG Pathway enrichment results showed that there were 18 signaling pathways involved in differentially expressed genes after HDR and LDR,which mainly included TGF-? signaling pathways,MAPK signaling pathways and metabolic pathways.There are 4 common pathways involved in the two groups of genes with opposite expression trends,namely MAPK signaling pathway,osteoclastdifferentiation,hematopoietic cell lineage,and systemic lupus erythematosus.(2)Based on the results of genomics and bioinformatics analysis,the effect of different doses of ionizing radiation on the expression of immune response-related cytokines and co-stimulatory molecules in mouse splenocytes was further examined by ELISA.The secretion of IFN? and IL-2,which have a positive effect on immune function in spleen cells.The expression of co-stimulatory molecules CD28 and CD40 L was increased.The secretion of IL-17,TGF-? and IL-21 with negative regulation was significantly down-regulated.The expression of co-stimulatory molecules CTLA4,ICOS,ICOSL,and CD40 was decreased.The secretion of cytokines and the expression of costimulatory molecules were reversed after HDR.(3)Further testing of related genes in the TGF-? signaling pathway revealed that low dose radiation can be induced in mouse thymocytes,spleen cells,EL-4,or in Jurkat cells(in vitro model of T lymphocytes)The expression levels of TGF-? gene and protein were decreased,while the expression of USP15 was significantly increased.However,after high-dose ionizing radiation,the expression levels of TGF-?gene and protein were significantly increased,while the expression of USP15 was significantly decreased.It can be seen that high-and low-dose ionizing radiation play an opposite role in the TGF-?/Smad7 signaling pathway of Jurkat cells,that is,low-dose radiation plays an inhibitory role,and high-dose radiation plays a promoting role.Further,in vitro inhibition of the key gene USP15 in the pathway revealed that USP15 has different mechanisms of regulation in different doses of ionizing radiation-induced TGF?/Smad7 signaling pathway.3.Effects of different doses of radiation on epigenetic changes in mouse thymocytesAccording to the results of whole gene expression profiling,it was found that some epigenetic related genes were differentially expressed in mouse immune cells after irradiation at high and low doses.Therefore,the molecular mechanism of epigenetics was investigated in this study.(1)Discovered in the study of the mechanism of action of epigenetic modifications related genes: DNMT-1 and EZH2 genes in the thymus of mice showed opposite changes after irradiation at high and low doses,that is,EZH2 gene expression was elevated after low dose irradiation,whereas DNMT-1 gene was highdose After irradiation,the expression increased;the change trends of Prc-1 and Paxip genes were the same after high dose irradiation and low dose irradiation,that is,the expression of the former increased and the latter decreased.(2)A total of 499 Lnc RNAs and 649 m RNAs were differentially expressed using the Lnc Path TM gene chip assay.Under high-dose ionizing radiation,36 Lnc RNAs were up-regulated and 17 Lnc RNAs were down-regulated compared to the control group.In addition,39 m RNAs were up-regulated and 35 were down-regulated.Only one Lnc RNA was significantly reduced in mouse thymocytes compared to the non-irradiated group after exposure to low dose ionizing radiation.The q RT-PCR method was used to verify the gene chip results.Three co-expression pathways(Lnc RNA: AK042609,EMSMUST0000058706,AK083162 and their target encoding genes: Ptpra,Hs3st11,Mbd2)were selected from low-dose irradiation groups and irradiated from high doses.Three pathways(Lnc RNA: AA089093,AK0432271,uc008 ref.2 and their target encoding genes: EZH2,Hells,Asf1a)were selected for verification in the group,and the results were consistent with the results of gene chip detection(p<0.05).The GO analysis of high-dose irradiation group found that down-regulated genes are mainly involved in biological processes such as chromatin remodeling,chromatin binding and chromatin-DNA binding.Up-regulated genes are mainly involved in target cell death,cell cycle progression,and programmed cell death.And other biological processes.It is also found that epigenetic modification related gene EZH2 plays an important role in the immune response induced by high and low dose radiation,and is related to the activation of the downstream transcription factor NF-?B.Conclution:1.Low-dose ionizing radiation induces differentiation of mouse lymphocytes into Th1 cells and induces a Th1-type immune response.However,high-dose ionizing radiation is involved in the differentiation of Th3/Tr1 cells and induces a Th2-type immune response.2.High and low doses of ionizing radiation cause differential expression of some genes in spleen cells of mice,and the different immune effects caused by the difference are mainly reflected in: DNA methylation,ubiquitination,protein-DNA complex recombination,DNA modification and cells Immunity and other aspects of regulation;3.Different doses of radiation induced different immune responses may be related to low dose radiation to enhance the positive regulation of cytokines and co-stimulatory molecule expression,and high dose radiation can enhance the negative regulation of cytokines and co-stimulatory molecule expression;4.TGF-?/Smad7 signaling pathway plays an important role in high and low doses of radiation-induced immune responses,and changes in TGF-? may be reversed by USP15;5.High-and low-dose radiation induced DNA methylation in immune cells,and the expression of regulatory genes involved in the process was not completely identical.EZH2 gene plays an important regulatory role;6.Lnc RNAs and their target gene networks may regulate biological processes such as cell proliferation and apoptosis,chromatin remodeling or repair,and cell macromolecule synthesis.Among them,AA089093 and EZH2 play important roles in different doses of ionizing radiation-induced immune responses. |
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