| Autoimmune diseases are often affected by the interaction of genetic and environmental factors.Ankylosing spondylitis(AS)is one of the most common genetic autoimmune diseases,which often affected young men aged 20-40.The clinical manifestations of AS are mainly spinal and sacroiliac arthritis,which can cause inflammatory osteopenia and abnormal hyperosteogeny.It can also contribute to acute uveitis,peripheral arthritis,tendinitis,aortic root disease and intestinal inflammation.The continuous evolution of the disease often leads to chronic pain,joint stiffness,limited chest expansion,limited spinal activity and even disability,which seriously affect the quality of life of patients and culminate in physical or mental pressure and economic burden.The incidence rate of AS in identical twins and familial aggregation study showed that the genetic function was more than 90%.For a long time,genetic research has provided convinced insights into the etiology and treatment of AS.Autoimmune diseases not only have susceptible gene variation,the change of immune system is also important to pathogenesis.Immune dysfunction is one of the earliest disease mechanisms proposed in AS.Clonal expansion of T cells to recognize target antigens to enhance adaptive immune response is an important feature of the disease.Immunological research involving reactive T cell population is helpful to explain the pathogenesis of AS and get fresh treatment.Together with genetic research,it helps to solve the problems of lacking of early diagnosis methods and insufficient treatment options of AS.T lymphocytes are considered to be the key cells in the regulation of the immune system.The number of CD4~+T cells and CD8~+T cells can be detected in the joint fluid of AS patients with early sacroiliac arthritis.In the peripheral blood of patients with AS,there is dysfunction of T cell subsets,mainly including the increase of the proportion of Th1 and Th17 cells,while the proportion of Treg and other cells is relatively reduced,and there is also TNF-α、IFN-γand IL-17.All these indicate that T cells play an important role in the pathogenesis of AS.CD247(ie CD3ζ、CD3zeta chain)is an important component of TCR/CD3 complex on the surface of T cells and participates in the transmission of the first signal in the process of T cell activation.Our study aimed to analyze the expression of CD247 in T cells of patients with AS,then to find the factors affecting the expression of CD247,further exploring its role in the pathogenesis.Also showing the possible clinical value,and providing a certain reference for the diagnosis and treatment of AS.Chapter I Analysis and verification of differential genes in ankylosing spondylitisObjective:to screen out the possible hub genes by analyzing the differential genes between as patients and healthy controls in the data set of GEO database,and design experiments for research and verification.Methods:1.Download and analyze the as related expression profile chip data set(GSE25101 and GSE73754 differential genes)in GEO database,and screen the genes with up-regulated expression or down-regulated expression in the two sets of data sets at the same time;2.The differential genes were analyzed by protein protein interaction network(PPI network),and the target genes of this experimental study were preliminarily screened;3.The expression of the screened target gene in the corresponding target cells was analyzed by flow cytometry;4.Design primers and detect the expression level of target gene m RNA by RT q PCR.Results:1.A total of 74 differentially expressed genes were screened in GSE25101 and GSE75735 data sets(including 41 up-regulated genes and 33 down-regulated genes);2.According to the results of PPI network analysis and previous research results,CD247 was selected as the target gene of this study;3.Flow cytometry showed that the expression of CD247 in T cells decreased in patients with AS,but no significant change in the expression of CD247 in NK cells.4.Compared with the healthy control group,the expression level of CD247 m RNA in peripheral blood T cells of as patients decreased significantly;Conclusion:through the screening of differential genes,CD247 was obtained as the target gene of this study,and the down-regulation of CD247 expression in peripheral blood T cells of patients with as was verified at the gene level and protein level.Chapter II Intracellular and extracellular factors affecting the expression of CD247 on T cellsObjective:by comparing the expression of CD247 in T cells stimulated by different inflammatory factors and different time conditions,to explore the possible extracellular environmental factors causing the change of CD247 expression and the possible mechanism of down-regulation of intracellular expression.Methods:1.The serum of AS patients and healthy controls were cultured in PBMC,and then the expression of CD247 in T cells was analyzed;2.Compare the serum cytokines between as patients and healthy controls,and analyze whether there is a certain correlation between them and T cell CD247;3.The expression of CD247 in T cells was detected by stimulating PBMC with related cytokines for 5 days;4.Peripheral blood PBMC of as patients and healthy controls were stimulated with different concentrations of Caspase-3 to detect the expression of CD247 in T cells.5.Design primers and detect the expression of LC3B m RNA in T cells by RT-q PCRResults:1 There are some cytokines in the serum of patients with AS,which can reduce the expression of CD247 in T cells;2.The levels of IL-2、IL-4 and IL-10 in serum of patients with AS were decreased,and they were not correlated with the expression of CD247 in T cells of patients with AS,but the expression of IL-6 and TNF-αis significantly increased,and negatively correlated with the expression of CD247 in T cells of AS patients;3.Under long-term stimulation,IL-2 can increase the expression of CD247 in T cells of AS patients.TNF-αcan down-regulate the expression of CD247 in T cells of healthy people and AS patients;4.Caspase-3 inhibitors can decrease the expression of CD247 in T cells,and Caspase-3 down-regulation may be an intracellular factor responsible for the down-regulation of CD247 expression.5.Compared with the healthy control,the expression of LC3B m RNA in T cells of AS patients was increased,indicating that the autophagy level was increased.Conclusion:Some inflammatory factors in serum of AS patients can cause changes in CD247 on T cells,IL-2 can increase the expression of CD247 on T cells,while TNF-αcan decrease the expression of CD247 on T cells,and it is speculated that the changes in the expression of CD247 in T cells may be affected by chronic inflammatory stimulation.Caspase-3 inhibitors can cause a decrease in the expression of CD247 in T cells.Based on this,Caspase-3 inhibition and its related intracellular pathways are hypothesized to be an intracellular factor of low CD247 in T cells.Chapter III Clinical significance of CD247 expression in T cellsObjective:to analyze the possible clinical significance by analyzing the correlation between the expression of CD247 in T cells and the indexes related to as disease activity.Methods:1.according to the expression of ESR and CRP in serum,patients with as were divided into ESR elevated group and ESR normal group,CRP elevated group and CRP normal group.The difference of CD247 expression between the two groups was compared and statistically analyzed;2.compare the correlation between the expression of CD247 and Bath Ankylosing Spondylitis Disease Activity Index(BASDAI),Bath Ankylosing Spondylitis function index(BASFI),Bath Ankylosing Spondylitis Metrology Index(BASMI)and Ankylosing Spondylitis Disease Activity Score(ASDAS)in patients with AS,and judge whether there is correlation between the expression of CD247 and disease activity in patients with AS;3.Statistically analyze the correlation between the expression of CD247 in T cells and CRP/Alb,and explain the possible relationship between the expression of CD247 in T cells and chronic inflammation.Results:1.There was no significant difference in the expression of T cell CD247 between ESR elevated group and ESR normal group,and between CRP elevated group and CRP normal group;2.There was no correlation between the expression of CD247 on T cells and BASDAI、BASFI、BASMI and ASDAS in patients with AS;3.There was a negative correlation between the expression of CD247 on T cells and CRP/Alb in patients with as.Conclusion:there is a certain correlation between the expression of CD247 on T cells and CRP/Alb in patients with AS.It is suggested that the expression of CD247 in peripheral blood T cells can reflect the systemic chronic inflammation of patients,which has certain clinical guiding significance,and provides a new reference for evaluating the severity of as patients. |
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