| Ankylosing spondylitis(AS) is a chronically inflammatory disease,which the pathogenic mechanism has not been clarified yet.The incidence of disease is about three in a thousand,which is a little bit inferior to rheumatoid arthritis.AS has obvious familial aggregation phenomenon which is related to HLA-B27 tightly.AS majorly affects axial skeleton,and its major characters are sacroiliitis which involved synovial joint,gristle joint and entheses that cause fibrosis and bone ankylosis often.Some other organs could be affected as well,for example,25%～30%patients may appear anterior uveitis.AS has very high disability rate,which brings extraordinary heavy psychological and economical burden.It is well known that HLA-B27 is the key susceptibility gene of AS.With the twins and familiy investigation we can see that this disease is oligo-genotype,and less than 50% part in the heritability can be explained by HLA-B27.Some report shows the polymorphism of IL-23R gene is highly related to AS,because it has 12%affection in the attributable risk in the patients group.Although the pathogenic mechanism has not been clarified,the common sense is the imbalance of immune system in the AS patient that has tight relationship with its pathology and clinic,in which the abnormity of T cell immune system plays an important role on its onset.More and more evidences show that the characters of cell excretion also affect the clinic process of spinal disease.Th17 cell is a helper T cell which is found in the recent years.All its characters are different from Th1 and Th2,and it produces cytokines include IL-17,IL-21å’ŒIL-22,etc.Some research proved that Th17 cell plays a vital roll in the pathogenic mechanism of autoimmune disease,such as experimental autoimmune encephalomyelitis,and inflammatory bowel disease and the polarization and functions are adjusted by different cytokines.IL-23R is a key gene to Th17 cell.The research will identify IL-23R gene variant or areas that related to AS in Han nationalities in China.At the same time,whether the IL-23R gene variant may affect Th17 cell's amount and functions and then cause AS will also be researched.The details are shown as the following three parts:Partâ… :Research of single nucleotide porlimorphism in the Chinese group of Ankylosing Spondylitis.Objective:8 SNP sites(rs11209026,rs1004819,rs10489629,rs11465804,rs1343151,rs10889677,rs11209032,rs1495965) of IL-23R will be inspected in the Chinese group.The purpose is to get the relationship of IL-23R single nucleotide porlimorphism with AS affectability.Patients and Methods:Refer to 1984 amended New York standard.109 AS patients are collected from clinic and in-patients,normal collators are 150 samples.8 SNP sites of IL-23R will be inspected by TagMan MGB probe methods on the patients.Porlimorphism, its hyploit,AS relative dangerous rate and date analysis are implemented by SPSS sys.Results:IL-23R SNPs related 8 sites rs11209026,rs1004819,rs10489629,rs11465804,rs1343151,rs10889677,rs11209032,rs1495965) has obvious correlation with AS in Chinese(P＜0.001).The strongest pertinent SNP is rs11209032(OR 1.3,95% CI 1.2-1.4,P=3.5×10-8).According to this gene related to the analysis of AS clinic,we find rs11209032A/G gene,rs11209032A/A gene,and rs11209032G/G gene in the patients of AS stands separately are 62 samples(56.1%),32 samples(29.4%),and 15 samples(13.8%).Regular comparison group stands separately are 71 samples(47.3%),45 samples(30%),and 34 samples(22.7%).No obvious difference between these two groups (P＞0.05).Partâ…¡:Th17 cell related cytokines inspection and functions initial discuss1.Inspection of Th17 cell related cytokinesObjective:Through inspecting IL-17,IL-23,IL-1β,IL-6,and TGFβ-1 level in the peripheral blood,identify Th17 cell related cytokines difference between AS patient and normal people and between different levels AS patients,in order to find out the effectiveness of Th17 cell in the AS pathogenic mechanism.Patients and Methods:Refer to 1984 amended New York standard.87 AS patients are collected from clinic and in-patients,normal collators are 91 samples.ELISA and Real-timer RT-PCR are used for inspection of IL-17,IL-23,IL-1β,IL-6,and TGFβ-1 from protein level and mRNA level to AS patients.AS patients are separated into two groups based on BASDAI≤4 in-active period and BASDAI＞4 active period for a comparison of all the cytokine level in AS patients.Independent sample t analysis and SPSS11.5 statistic software are used for statistic and analysis.Results:â‘ IL-17 protein level and mRNA level of AS patients is much higher than normal control group(75.45±6.46 vs 41.97±2.27,P＜0.001;0.003240±0.000872 vs 0.007643±0.001656,P＜0.05);AS patients are separated into two groups based on BASDAI≤4 in-active period and BASDAI＞4 active period,for the comparison of IL-17 mRNA level between AS patients.IL-17mRNA level of active period AS patients is higher than in-active patients(0.001213±0.000765 vs 0.004561±0.001404,P＜0.05).â‘¡IL-23 proteinlevel and mRNA level of AS patients is much higher than normal control group (61.85±2.61 vs 54.45±2.53,P＜0.05;0.038562±0.013772 vs 0.078789±0.014030, P＜0.05);Classified comparison of IL-23mRNA level of AS patients shows,IL-23mRNA level of active period AS patients is higher than in-active patients(0.010229±0.001813 vs 0.0679466±0.027626,P＜0.05).â‘¢IL-1βprotein level and mRNA level of AS patients is much higher than normal control group(28.45±0.866 vs 22.25±0.603,P＜0.001; 0.324742±0.128426 vs 1.426351±0.464371,P＜0.05);Classified comparison of IL-1βmRNA level of AS patients shows,no obvious difference between active period and in-active patients(P＞0.05).â‘£IL-6 protein level and mRNA level of AS patients is much higher than normal control group(5.10±0.52 vs 2.79±0.31,P＜0.001;0.013524±0.005065 vs 0.058685±0.015024,P＜0.05);Classified comparison of IL-6 mRNA level of AS patients shows,IL-6 mRNA level of active period AS patients is higher than in-active AS patients (0.003831±0.001475 vs 0.044567±0.013595,P＜0.05).⑤TGFβ-1 protein level and mRNA level of AS patients is much higher than normal control group(14.09±1.76 vs 3.59±0.60,P＜0.001;0.200738±0.048957 vs 1.098782±0.318909,P＜0.05);Classified comparison of TGFβ-1 mRNA level of AS patients shows,TGFβ-1 mRNA level of active period AS patients is higher than in-active AS patients(0.058109±0.017780 vs 0.256579±0.075089,P＜0.05).Conclusions:These results show that cell gene level of IL-17,IL-23,IL-1β,IL-6, and TGFβ-1 in the peripheral blood of AS patients is higher than normal controls.That is to say,Th17 cell related cytokines should be working in the AS pathogenic mechanism.2.Th17 cell function initial discussionObjective:Inspect Th17 cell distribution status of AS patients and normal controls and make comparison.At the same time,observe the affection of different cytokines stimulation to Th17 polarization,and discuss the difference of Th17 cell functions between AS patients and normal people.Patients and Methods:Collect 10 AS patients,and 15 normal collators.Ficoll desity gradient centrifugation methods is used for isolating PBMC first,then five hours stimulation has to be implemented by phorbol ester and calcium ionophore,anti CD4-FITC, anti IL-17-PE,anti CD8-FITC,and anti IL-17-PE has to be marked,and the rate of CD4+IL-17+T cell and CD8+IL-17+T cell has to be inspected by Flow Cytometry.Part of PBMC should be added in different cell gene IL-1β+IL-6,IL-1β+IL23,IL-6+IL23, IL-1β+IL23+IL-6 and should be maintained for five days,anti CD4-FITC and anti IL-17-PE should also be marked,and the relative contents should be inspected by Flow Cytometry.Results:â‘ CD4+IL-17+ lymphocyte in the peripheral blood of AS patients is higher than normal controls(2.79±0.20 vs 0.06±0.01 P＜0.01),and IL-17 are mainly excreted by CD4+T lymphocyte,but CD8+T lymphocytes do not excrete much.â‘¡after the stimulation of different cell gene to T cell,the results show IL-1βand IL-6 cell gene have obvious result to the stimulation of IL-17.Conclusions:Peripheral blood IL-17+ and T cell peripheral blood distribution of AS patients are higher than normal collators group,and CD4+IL-17+ cell group is the majority group.IL-1βand IL-6 may effectively stimulate Th17 cell polarizeation,and lead to the appearance of IL-17. |
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