| Objective: To study the mechanism of Th17 cel s differentiation induced by superantigen SEA in peripheral blood,according to the role of Th17 cells in the development of autoimmune disease and the research on T cells in response to staphylococcus exotoxin A stimulation,which will provide the experimental basis on the pathogenesis of autoimmune diseases such as RA.Methods: 1.To measure the levels of cytokines IL-17A、IL-1β、TGF-β1、IL-6、IL-23 or IL-21 secretion by ELISA.2.To detect the levels of STAT3 or RORγt m RNA expression in PBMC by Real Time-PCR.3.To detect the levels of STAT3 or RORγt protein expression in PBMC by Western blotting.4.To analysis the percentage of Th17 cells in PBMC by flow cytometryResults: 1.The secretion of cytokines IL-17A、IL-1β or IL-23 was enhanced along with the increase of SEA concentration(0、5、10、50、100、500ng/ml)(P<0.05).However,the secretion of IL-6 or TGF-β1 didn’t depend on the concentration of SEA(P>0.05).The level of IL-21 secretion was low,and beyond the detection sensitivity of ELISA kits.2.The secretion of cytokines IL-17A、IL-1β or IL-23 was up-regulated over time(0、6、12、24、48、72h)(P<0.05),when PBMC were stimulated with 500ng/ml SEA.Also,the secretion of IL-6 or TGF-β1 had no time-effect relationship with SEA(P>0.05),but the levels of IL-6 was high,and increased in the early stage of cytokines secretion.3.Compared with normal control group,the secretion of cytokines IL-17A、IL-1β or IL-23 were obviously enhanced,and in response to SEA、Anti-CD3/CD28、LPS stimulation(P<0.05).The expressions of IL-17 A 、IL-1β or IL-23 were significantly different among three groups(P<0.05),and the level of IL-17 A 、IL-1β or IL-23 expression in SEA or Anti-CD3/CD28 group was higher than that of LPS group(P<0.05);However,the level of TGF-β1 expression in SEA group was higher than that in LPS group(P<0.05),it had no statistical differences between the other groups(P>0.05).Besides that,the differences of IL-6 secretion were not obvious different among all of groups(P>0.05).4.The expression of STAT3 or RORγt m RNA were obviously elevated in PBMC following SEA、Anti-CD3/CD28、LPS stimulation,and compared with normal control group(P<0.05).The expression of STAT3 or RORγt m RNA in SEA group was higher than that in LPS group(P<0.05),and that of Anti-CD3/CD28 group was highest.5.STAT3 protein and RORγt protein were increasingly expressed in PBMC stimulated by SEA、Anti-CD3/CD28 and LPS,and compared with normal control group(P<0.05).The expression of STAT3 or RORγt protein in SEA group and Anti-CD3/CD28 group were higher than that of LPS group(P<0.05).Furthermore,the expression of STAT3 or RORγt protein in SEA group had no statistical differences with that of Anti-CD3/CD28 group(P>0.05).6.Compared with SEA group、Anti-CD3/CD28 group and LPS group with adding the same dosage of IM,the expression of STAT3 or RORγt protein in SEA or Anti-CD3/CD28 group was inhibited;The expression of STAT3 protein of SEA group and LPS group increased slightly,compared with the negative control group(P<0.05);The level of STAT3 protein expression in Anti-CD3/CD28 group had no statistical differences with the negative control group(P>0.05).Meanwhile,compared with the negative control group,RORγt protein expression in SEA group and Anti-CD3/CD28 group were not enhanced(P>0.05),but that in LPS group was still up-regulated(P<0.05).7.The percentage of Th17 cells in SEA group、Anti-CD3/CD28 group and LPS group was higher than that of the normal control group(P<0.05).The percentage of Th17 cells in SEA group and Anti-CD3/CD28 group were more than that of LPS group(P<0.05),and the level of Th17 cel s differentiation in Anti-CD3/CD28 group were highest.Conclusions: 1.SEA promotes the secretion of cytokines IL-17 A,IL-1β and IL-23 in normal PBMC.2.IM inhibits Th17 cells differentiation induced by SEA.3.SEA may up-regulate the expression of STAT3 or RORγt at the levels of gene and protein via the TCR signaling pathway.4.SEA may induce Th17 cel s differentiation which is mainly in the form of superantigen via TCR pathway. |
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