| Autism,also known as autism spectrum disorder(ASD),is a pervasive neurodevelopmental disorder characterizaed by deficits in communication and social interactions,as well as the presence of stereotypic behaviors.In the United States,ASD may affect as many as 1 in 68 children,and in China,the incidence is about 1%,accounting for the most neuropsychiatric disorders in children.It is believed that the occurrence of autism is the result of a combination of genetic,environmental and biological factors.There are now more than 600 genes specifically associated with autism,such as FMR1,MECP2,nlgn3/4,NRXN1,SHANK3,tsc1/2,and PTEN.In the genome-wide association analysis(GWAS)of genes in the linkage region of human chromosome 1q13.2,professor Yang feng found that SORT1 gene was an autism susceptibility gene.JW Trampush et al found that SORT1 gene is a cognition-related functional gene,whose mutation leads to the change of cognitive function and is closely related to the occurrence of neurological diseases,degenerative diseases and autism.Arti B et al.confirmed that SORT1 gene encoding protein sotilin is highly expressed in autistic patients,and it can be involved in microglia pathological process and inhibit the expression of sortilin protein by activating m TOR pathway,which will contribute to the treatment of autism.Microglia cells are dormant macrophages in the central nervous system and are also innate immune cells,which play an important role in inflammation and immune response of the central nervous system.Chronic long-term activation of microglia is thought to be detrimental to neurons.A large amount of clinical data showed that the contents of IFN-?,il-1,il-6 and TNF-in serum of autistic children were significantly increased,and the contents of MCP-1,il-6,IFN-?,il-8,MIP-1 and other pro-inflammatory factors and regulatory cytokines in cerebrospinal fluid were significantly increased.The high expression of these inflammatory factors was closely related to the core symptoms of autism.NF-?B is an important nuclear transcription factor.NF-?B signaling pathway is involved in the regulation of physiological and pathological processes such as inflammation,innate immune response,and adaptive immunity.Pathological activation of NF-?B signaling pathway will lead to chronic inflammation,immune deficiency,brain degeneration and other diseases.New study has found that combining with P75 NTR sortilin can activate the NF-?B pathway thereby,based on previous work,discovers sortilin and inflammatory cytokines in peripheral blood in patients with autism is significantly higher than normal people,but whether sortilin the NF-?B pathway activation of microglia is involved in autism in development process,and did not see,therefore,this study based on the VPA autism rats model,observation sortilin expression in brain development,determine whether sortilin involved in autism in process,Then,from the perspective of microglia inflammatory response,the possible mechanism of sortilin involved in the regulation of autism was discussed.Part ? Expression of Sortilin in autism and VPA Model Rats Objective: To investigate the expression of sortilin in autism and VPA autism model rats Methods: 1.Sortilin in serum of 13 normal children and 11 autism aged 3-12 years was detected by ELISA.2.VPA was intraperitoneally injected into pregnant rats at embryonic day12.5 and their offspring were subjected to neurobehavioral test.Through three-box experiment,social experiment and open field experiment,the social communication ability of model rats was tested,and the stroking experiment was conducted to detect their rigid and repeated behaviors,so as to establish the VPA autistic rat model.3.Western blot method was used to detect the expression of sortilin at different time of hippocampal development in VPA rats.Results:1.ELISA results: Serum sortilin in autism is significantly higher than normal children.2.Western blot results: sortilin was higher in the whole early hippocampal development(P7D,P14 D,P35D)of VPA autistic model mice than in the normal group.Conclusion: Sotilin1 is significantly increased in serum of autistic children and in VPA brain tissue of autistic rats.Combined with the genome-wide association analysis(GWAS)results of human chromosome 1q13.2 linkage region gene,we believe that SORT1 gene is an autism susceptibility gene,and SORT1 protein sortilin is involved in the regulation of autism.Part ? Sortilin Activates the Mechanism of Inflammatory Response Involved in Autism Objective: to investigate whether sortilin is involved in inflammatory response and mechanism of autism Methods: 1.Western blot was used to detect the expression of IL-1 and IL-18 in the hippocampus of VPA model rats,and immunofluorescence staining was used to observe the number and morphology of microglia cells in the hippocampus of VPA model rats.2.To further verify the relationship between sortilin and inflammatory response,we used si RNA technology to specifically knock down the SORT1 gene in the hippocampus of VPA autistic rats,and then used immunofluorescence method to detect the changes in the number and morphology of small glia,and western blot to detect the expressions of IL-1 and IL-18.3.In order to investigate whether the inflammatory response can be induced by the production of inflammatory factors through the NF-?B signaling pathway,we used the fluorescence double label to detect the dynamic expression of p65 in the hippocampal brain tissue.Nuclear transcription factor NF-?B usually binds to its inhibitory protein I?B in an inactive state as a heterodimer of p50-p65.The activation of NF-?B pathway is shown as the dissociation of NF-? B.Therefore,we observed the dynamic changes of p65 in the cytoplasm and nucleus.Results:1.Western blot results: the expression of IL-1 and IL-18 in the hippocampus of VPA rats was significantly higher than that of the control group.Immunofluorescence results: the number of microglia cells and their protrusions were significantly higher than that of the control group.2.After specific knockout of the SORT1 gene in the hippocampus of VPA autistic rats,Iba-1 immunofluorescence showed that: compared with the non-knockout group,the number and activity of hippocampal microglia were significantly decreased.Western blot results: both il-1 and il-18 were reduced compared with the untreated group.3.P65 immunofluorescence observation: in1 the hippocampal tissue of VPA autistic rats,the expression of P65 in the cytoplasm and nucleus increased,and the nucleation of P65 was obvious.When specific knockout of the SORT1 gene in the hippocampus of VPA autistic rats can significantly inhibit the dissociation and nucleation of p65.Conclusions: 1.The core symptoms of autism may be associated with persistent microglia inflammatory responses.2.The high expression of sortilin in autism can trigger the inflammatory response of microglial cells in the hippocampus.3.Sortilin promotes the inflammatory response in the hippocampus through the NF-?B pathway.Part ? After the knockout of SORT1 gene,the autism-like behavior of VPA autism model rats were improved Objective: To investigate whether the autism-like behavior of VPA autism model rats was improved after SORT1 gene knockoutMethods: We performed ASD-related neurobehavioral tests on VPA rats with SORT1 gene knockout,including three box experiment,social experiment,open field experiment and stroking experiment.Results: Compared with the non-knockout group,specific knockout VPA autistic rats had significantly increased contact time with unfamiliar rats in three-box experiment,social experiment and open field experiment,increased exploration time of new environment,increased communication time between rats,and significantly reduced number of wool stroking experiments.Conclusion: Knockout of SORT1 gene can improve the social interaction ability of VPA autistic rats. |
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