| Objective:To explore the changes of Th17 cells associated factors in peripheral blood from patients with Graves disease(GD) and detect the expression levels of glucocorticoid-induced tumor necrosis factor receptor(GITR) mRNA in PBMC from patients with GD by real-time fluorescence quantitative PCR (QRT-PCR) method.Method:(1) The thyroid hormone(FT3,FT4 and TSH) levels in plasma from 22 untreated GD patients,23 GD patients in clinical remission and 18 healthy controls was measured by ICMA.(2) PBMCs from each group were extracted into total RNA,after they were stimulated for 5h by PMA(50ng/ml) and Ionomycin(1μg/ml).RORγt, IL-17 andβ-actin mRNA copies/μl in these total RNA were quantitated by QRT-PCR.β-actin gene was served as internal reference,and the ratios of RORγt,IL-17 toβ-actin mRNA represented their relative expression levels, and the correlations between the levels of RORγt,IL-17 mRNA and thyroid hormone in untreated GD patients were evaluated.The levels of IL-17 and IL-23 in plasma were detectd by ELSIA.(3) Total cellular RNA was extracted from human PBMC and hGITR gene obtained by RT-PCR was cloned into pMD18-T vector.The pMD18T -hGITR vector was transformed into E.coli DH5αand identified by restriction digestion,PCR and DNA sequencing,the recombinant plasmids including GITR gene were prepared.The method of QRT-PCR for the detection of this gene was established by the recombinant plasmids,which served as the Standard plasmids.The linear range,specificity and degree of precision of this method were evaluated.GITR andβ-actin mRNA copies/μl in PBMC were quantitated by this method and the ratio of GITR toβ-actin mRNA represented its relative expression,and the correlations were evaluated between the relative expression level of GITR mRNA and thyroid hormone levels in untreated GD group.Also the correlations were evaluated between GITR mRNA and RORγt and IL-17 mRNA in PBMC from untreated GD patients.Result:(1) The levels of FT3 and FT4 in plasmas from untreated GD patients were higher notedly than that from GD patients in clinical remission and healthy controls(P＜O.05),and the levels of TSH in plasmas from untreated GD patients were lower notedly than that from GD patients in clinical remission and healthy controls(P＜O.05).but there was no significant difference about the levels of the thyroid hormone between the groups of GD in clinical remission and healthy control(P＞0.05).(2) The relative expression level of RORγt mRNA in PBMC from 22 untreated GD patients was higher notedly than that from 23 GD patients in clinical remission(χ~2=11.76,P＜0.01) and 18 healthy controls(χ~2=6.62, P＜0.05),but there was no significant difference between the groups of GD in clinical remission and healthy control(χ~2=0.42,P＞0.05).Also the relative expression level of IL-17 mRNA in PBMC from untreated GD patients was higher notedly than that from of GD patients in clinical remission(χ~2=20.91, P＜0.01) and healthy controls(χ~2=26.92,P＜0.01),but there was no significant difference between the groups of GD in clinical remission and healthy control (χ~2=0.84,P＞0.05).The levels of IL-17 in plasma from untreated GD patients was higher notedly than that from GD patients in clinical remission(χ~2=26.67, P＜0.01) and healthy controls(χ~2=32.08,P＜0.01),but there was no significant difference between the groups of GD in clinical remission and healthy control (χ~2=0.68,P＞0.05).There both were positive correlations between the levels of RORγt,IL-17 mRNA and FT3,FT4 in untreated GD patients(P＜0.05),rather there both were negative correlations between the levels of RORγt,IL-17 mRNA and TSH in untreated GD patients(P＜0.05).Also the levels of IL-23 in blood plasma from untreated GD patients was higher notedly than that from GD patients in clinical remission(χ~2=23.91,P＜0.01 ) and healthy controls (χ~2=35.88,P＜0.01),but there was no significant difference between the groups of GD in clinical remission and healthy control(χ~2=2.01,P＞0.05).(3) The recombinant plasmids of pMD18T-hGITR including 103 bps of GITR gene by PCR,which shared 100%homology with the sequence of human GITR provided by the Genbank,was constructed successfully.As for this method,the correlation coefficient of the standard curve was 0.999,the linearity ranged from 10~1 copies/μl to 10~6 copies/μl,the sensitivity reached 10~1 copies/μl,the CV of the intra-assay was 6.4%,the CV of the interassay was 13.7%and 12.4%.The relative expression of GITR mRNA in PBMC from untreated GD patients was lower notedly than that from GD patients in clinical remission(χ~2 =18.48,P＜0.01 ) and healthy controls(χ~2 = 18.26,P＜0.01 ), but there was no significant difference between the groups of GD in clinical remission and healthy controls(χ~2=0.12,P＞0.05).As for these correlations, there were negative correlations between the levels of FT3(r =-0.509, p=0.015),FT4(r =-0.483,p=0.023) and GITR mRNA,and there was a positive correlation between the levels of TSH(r=0.458,p=0.032) and GITR mRNA in untreated GD group.Also there both were negative correlations between GITR mRNA and RORγt mRNA in untreated group(r=-0.475, p=0.009) and in healthy group(r=-0.436,p=0.011),and there both were negative correlations between the relative expression levels of GITR and IL-17 mRNA in untreated group(r=-0.536,p=0.002) and in healthy group(r=-0.492,p=0.008).Conclusion:(1) The results indicate that the levels of RORγt,IL-17 and IL23,which were associated with Th17 cells,increase in GD patients. (2) The QRT-PCR method for the detection of GITR mRNA is a sensitive,specific and repeatable method.The research results indicate that the relative expression level of GITR mRNA was low in GD patients. |
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