| Background and Objective: Multiple sclerosis(MS)is a typical chronic autoimmune disorder that featured as subsequent axon injury and inflammatory demyelinating in the central nervous system(CNS).The clinical manifestations include neuritis,ataxia,and paralysis.As a typical model of human multiple sclerosis,experimental autoimmune encephalomyelitis(EAE)is widely used to explore auto-immunology.Pin1 is a member of the peptidyl prolyl cis / trans isomerase subfamily of fine proteins,which has a profound influence on many key proteins' conformational regulation and many important cellular activities.Studies have shown that Pin1 plays an important role in the development of asthma and the immune system related diseases such as microbial infection.However,the functions and mechanisms of Pin1 in autoimmune diseases have not been reported.The study is aimed to explore the effects of Pin1 on EAE in vitro and in vivo,reveal the molecular mechanisms,target the regulation of Pin1 on EAE,and provide theoretical basis for the treatment of multiple sclerosis and T cells related-immune inflammatory neurodegenerative disease.Methods: This thesis were studied mainly from the following three aspects: the overall EAE mice and the pathological analysis,the regulation effect of Pin1 on T cell subgroup in the spinal cord and peripheral lymph nodes of EAE mice,and in vitro study of the pathogenic effect of Pin1 on EAE mice.First we build EAE animal models in mice,and the 20 mice were divided into two groups,each group of 10.The experimental group mice were injected recombinant lentivirus vector by tail intravenous used to knockdown Pin1 a week before inducing.The control group mice were injected blanklentivirus vector by tail intravenous a week before inducing.After immune induction,the observation record was started and the clinical score was timely.We collected the spinal cord of EAE mice,and stained with HE staining and Luxol Fast Blue to detect the degree of inflammatory infiltration of the spinal cord and demyelination of the spinal cord.Then we charged fresh spinal cord and peripheral lymph nodes of EAE mice,obtained the mononuclear cells,the infiltration and proportion of T lymphocyte subgroups in the spinal cord and peripheral lymph nodes were measured by flow cytometry.Finally,we researched on the pathogenic effect of Pin1 on EAE in vitro.We collected the fresh spleen and lymphoid tissue of the EAE mice and isolate the individual nuclear cells.The cell proliferation and thesurface expression of DC were detected by flow cytometry.We used the dendritic cell lines and lymphocyte lines of Pin1 were knocked down in vitro.Reverse transcription of RNA,fluorescence quantitative PCR was used to study the expression of inflammatory factors and the expression of Th17 related genes.Results: 1.The changes of clinicopathological grading of EAE mice after Pin1 were knocked down.Compared with the control group,knockdown of Pin1 inhibited the development of EAE.The onset time of EAE with Pin1 knockdown was slowed 2days,and the trend of disease onset tended to be gentle.The score results were significantly lower than that of the control group.By statistics of the highest average scores and the average total score of each treatment group,we found that the total score and the average highest score of clinical score of EAE knockdown mice decreased significantly,and the incidence rate of Pin1 mice was also significantly lower than that of the control group.2.The degree of inflammation of the spinal cord and the degree of demyelination.At the peak stage of the disease,HE staining of paraffin sections of lumbar segments showed that inflammatory cells accumulated in the white matter of the spinal cord.White matter was mainly composed of nerve axons responsible for nerve conduction,suggesting that the axons were partly infiltrated by inflammatory cells.Paraffin section HE staining can observe the degree of inflammatory infiltration in the central nervous system,while solid blue staining specifically combined with myelin sheath.Therefore,it is used as a staining method to observe demyelination in nervous system.The experimental results showed that the infiltration of some inflammatory cells in the spinal cord of EAE mice with Pin1 knockdown was reduced,while the control group had obvious inflammatory cell clusters,forming dark patches,and mostly distributed in the white matter of the spinal cord.At the same time,blue staining also found that the infiltration area of the inflammatory cells was close to the demyelinating region of the nerve.In the control group,the infiltration of inflammatory cells in the white matter was high,and in the white matter,the demyelination part was particularly prominent in the white matter.The loss of Pin1 demyelinating flaky blue deletion was significantly reduced.3.Knockdown Pin1 inhibited the differentiation of Th17/Th1 subgroups of the lymphoid tissue effector T cells in EAE mice.The ratio of Th17 and Th1 cells in peripheral lymph nodes of mice was significantly reduced by knockdown Pin1.Further,the cells in the central nervous system(spinal cord)were detected by flow cytometry,and it was found that knockdown Pin1 could significantly reduce Th17 proportion in mice spinal cord.4.Knockdown Pin1 can inhibit the ripening and differentiation of dendritic cell DC.After knockdown Pin1,the DC of the EAE mice showed decreased ability of specific antigen presentation,and the corresponding CD83 and MHC-II expression were significantly reduced.The expression level of pro-inflammatory cytokines Il-6and Il-1 in DC cell lines decreased,while the expression level of anti-inflammatory factor Il-10 increased.5.Knockdown Pin1 can reduce the activation of T lymphocytes and the expression of related inflammatory genes.In mice with low Pin1,the value of T cells in the lymph nodes of the lymph node was significantly reduced in the value of specific antigen stimulation,and its function was significantly inhibited.In T cell line EL4 cells,knockdown Pin1 significantly decreased the expression level of the gene Il-17 A,Il-17 F and STAT3 associated with Th17.Conclusions: Knockdown Pin1 reduced the degree of inflammatory cell infiltration in the spinal cord of EAE mice,improved demyelination of EAE mice;knockdown of Pin1 inhibited the development of EAE;Pin1 silencing could inhibit the activity of EAE,it can be used as a target for the treatment of EAE.Knockdown Pin1 can significantly reduce the Th17 ratio in peripheral lymph nodes of mice;knockdown Pin1 can significantly reduce the Th1 ratio in peripheral lymph nodes of mice.The regulation effect of Pin1 on EAE and the regulation of autoimmune inflammation in EAE were confirmed.In conclusion,knockdown Pin1 can effectively improve the experimental mouse encephalomyelitis.Pin1 can affect the cell differentiation of the Th cell by regulating the DC,and it can directly affect the differentiation of Th cells.Pin1 to Th17 cells and its mediated autoimmune inflammatory reactions in EAE adjustment results,for a comprehensive understanding of MS/EAE pathological inflammatory diseases in the nervous system is of great significance,is expected to become the new target for diagnosis and treatment of related diseases. |
PDF Full Text Request