High Inorganic Phosphorus Intake Interferes With Immune System Through Decreasing Murine T Cell Levels

1 ObjectiveThis thesis was based on the previous experimental research.We used transcriptase technique to analyze the effect of inorganic phosphate on gene expression level in mice.By looking for genes that were significantly different from the control group,we determined the direction of research and made a further discussion on the mechanism.The test was based on the transcript analysis.It was further confirmed that inorganic phosphorus could cause the change of CXCL11 gene expression by using q-RTPCR technique.To explore the effects of high inorganic phosphorus uptake on the immune system from a new point of view,We focused on the correlation between T lymphocytes that secreted CXCL11 and inorganic phosphorus uptake level.2 Methods(1)The SPF BALB/c female mice were fed with inorganic phosphate food additives for 15 consecutive days,and LPS was used to stimulate mice to establish inflammation model.And observed the effect of inorganic phosphate intake on inflammatory response in mice.(2)Flow cytometry was used to detect the percentage of T lymphocyte in CD28~+,CD28~-cells and the ratio of Ts/Tc.And we analysed the effect of inorganic phosphates on the differentiation of T lymphocyte subsets.(3)Used the high-throughput transcriptome assay to analyse the effects of high inorganic phosphorus diet on immune system and gene expression related to tumor progression in mice.To explore the significant pathways of immune function in mice by high inorganic phosphorus diet,and find the differential genes.(4)After finding out the differential genes from RNA-Seq experiments,these genes needed to be tested.Thus,we can verify whether the high inorganic phosphorus diet can interfere with the immune system of mice and analyzed it.3 Result(1)Animal experiments showed that there was no significant difference in physiological activity of mice between this 5 groups during 15 days of intragastric administration.When LPS was injected,the mice in LPS group and phosphate dose group were unhealthy and their food intake decreased significantly.It indicated that the inflammatory model was successfully established,and the analysis was carried out through the further experiments.(2)The flow cytometry experiments showed that when the concentration of inorganic phosphate food additives that were administered to the stomach is 100mg/kg,200 mg/kg and 400 mg/kg,compared with LPS group,with the increasing concentration of inorganic phosphate food additives,the proportion of CD4~+and CD3~+decreased gradually.The changes of T lymphocyte subsets such as CD25~+,comprehensive analysis.(3)The transcriptome sequencing experiments showed that after a series of tests,reliable data were obtained,which enriched the information of sample database.From the biological point of view,we can see that,to some extent,the GO data showed that inorganic phosphates did affect the spleen function of mice,such as immune response,chemokines related to inflammation.In the KEGG Pathway section,we selected two immune significant pathways for further analysis.They were Toll-like Receptors pathway and HIF-1 signaling pathway.Compare with the group of LPS,three differential genes,such as STAT1,STAT3 and CXCL11 were found.The expression of STAT1 and CXCL11 was down-regulated,while there was no significant difference of STAT3.(4)The experiment of q-RTPCR showed that the verification results of STAT1,STAT3 and CXCL11 genes were consistent with the results of transcriptome sequencing.It provided a theoretical reference for further studying the effects of inorganic phosphate food additives on immune system and tumorigenesis in mice.4 ConclusionIn this study,flow cytometry was used to detect T lymphocyte subsets in immune-related thymic organs.It was verified that the balance of T lymphocyte population in mice was destroyed and the level of immune system in mice was declining.Meanwhile,high-throughput sequencing was used to detect the transcriptome data of the immune organ.The data resources were abundant.The hypothesis that inorganic phosphate food additives have immunosuppressive effect on BALB/c mice was validated.Significant immune-related pathways were preliminarily found out.It laid a solid foundation for further study on the mechanism of the effect of inorganic phosphate on immune mechanism.