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Objective To investigate the promoter methylation of drug metabolism genes CYP1A1 and CYP1B1 in thyroid cancer and its relationship with clinical pathological characteristics. Method 201 cases of thyroid cancer and 23 cases of normal thyroid tissues were involved. Methylation-specific PCR ( MSP ) was performed to analyze promoter methylation of CYP1A1 and CYP1B1 genes in the above tissues to detect the frequency of methylation positive, compare the promoter methylation level of CYP1A1 and CYP1B1 genes in papillary thyroid carcinomas ( PTC) and the controls. Five thyroid cancer cell lines were treated with methyltransferase inhibitor 5-Aza-dC for 5 days, and real time PCR ( RT-PCR) was performed to evaluate the mRNA expression of CYP1A1 and CYP1B1 genes. Logistic regression was used to analyze the correlation between the aberrant methylation and the clinical features. Results Aberrant methylation status in promoter region of CYP1A1 and CYP1B1 genes were detected in all kinds of thyroid cancers. Compared with control tissues, the methylation in promoter regions of CYP1A1 in PTCs was significantly higher, while that in promoter regions of CYP1B1 was lower (P<0.05). In vitro, 5-Aza-dC treatment significantly increased the CYP1A1 gene mRNA expression for 6. 92 and 8. 30 times in K1 and C643 cell lines respectively and restored CYP1B1 gene mRNA expression for 7.62 times in K1 cell line. Compared with the controls, PTCs with methylation in promoter regions of CYP1B1 had decreased lymphatic metastasis rate. Conclusion The methylation in promoter regions of CYP1A1 and CYP1B1 gene may regulate their mRNA expressions. Aberrant methylation of the promoter region of CYP1B1 is associated with lymph node metastasis in PTC.
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Objective To investigate the feasibility of inducing neonatal immunotolerance against Graves'disease by gene TSH receptor (TSHR) 289 and its possible mechanism.Methods Neonatal (0-24 h) female BALB/c mice were divided into intraperitoneal injection group,intramuscular injection group,model group,and normal control group.The intraperitoneal group and the intramuscular group were further divided into low-dosage,middle-dosage,high-dosage tolerance groups,and the coresponding control groups.The tolerance groups and the controls were intraperitoneally or intramuscularly pretreated with low-dosage( 1×106 particles),middle-dosage( 1 × 108particles),high-dosage( 1 × 1010 particles)of Ad-TSHR 289 or Ad-lacz respectively.6 to 7 weeks later,the normal control group received intramuscular injection with Ad-lacz; the other groups were immunized with Ad-TSHR289,three times at 3 weeks interval.10 days after the first immunization,serum TRAb was detected.4 weeks after the last immunization,serum TRAb,TT4,splenic CD4 + CD25 + Foxp3/CD4 + were tested,and the thyroid tissues were examinated histologically.Results Ten days after the first immunization,no antibody response against TSHR was detected in the two high-dose tolerance groups,but the TRAb titer in respective controls was significantly higher( P＜0.05 ).4 weeks after the last injection,in high-dose tolerance groups,only 1/10 of mice immunized by intraperitoneal or intramuscular injection elicited anti-TSHR antibody,and no mice immunized intraperitoneally had elevated serum TT4.Two of ten mice challenged intramuscularly showed slightly increased TT4 levels,but the respective controls displayed a strong antibody response( P＜0.01 ) and elevated TT4 level ( P＜0.05 ).The similar percentages of high TT4 and thyroid hyperplasia were found in all groups.Additionally,the frequencies of CD4+CD25 +Foxp3/CD4+in two high-dose tolerance groups were significantly increased as compared to those in controls( P＜0.05 ).The incidence of Graves' disease in the other groups by intraperitoneal or intranuscular injections was not statistically different from those in the corresponding control groups and the model group.Conclusions The immune tolerance against Graves'disease is induced in neonatal mice by either intraperitoneal or intramuscular pathway with specific antigen of TSHR 289,carried by adenovirus vector,and then inhibits Graves' disease in adults. Stimulation with the high-dosage antigen is liable to induce immune unresponsiveness.CD4 + CD25 + Foxp3 +T cells may play an important role in the induction and maintenance of tolerance.
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Objective To investigate the effect of mice gender on the TSH receptor antibody(TRAb)titers, the levels of TT4,and the degree of thyroid hyperplasia by establishing an animal model of Graves′ disease in male and female BALB/c mice. Methods Male and female BALB/c mice were immunized with recombinant adenovirus expressing TSHRA subunit(Ad-TSHR289)to induce Graves′ disease. Animals were injected 3 times at intervals of 3 weeks. All mice were sacrificed 4 weeks after the last injection to obtain blood for measurement of TSHR antibody titers and TT4evels, and thyroid glands for histological examination. Results TRAb positive rates were 100% both in female or male mice. No significant difference was observed in titers of TRAb between them. The incidence of hyperthyroidism in female mice was higher than that in male mice, being 75.0% and 41.7% respectively. There was statistical difference in levels of TT4between females and males(P＜0.01). Mice with high TT4exihibited marked thyroid hyperplasia. Conclusion Despite TSHR antibodies were similar between female and male mice, the incidence and degree of hyperthyroidism showed sex bias in Graves′s animal model. The results indicated that it was easier to induce model in females than in males by immunizing BALB/c mice with Ad-TSHR289.
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Objective To investigate the incidence and the possible risk factors of hepatic dysfunction in patients with Graves' disease (GD). Methods Totally 280 outpatients with hyperthyroidism (caused by GD) were divided in two groups: untreated group (131 patients) and treated group (149 patients). The hepatic function, serum thyroid hormone, TGAb and TMAb were detected for analysis of the incidence, clinical characteristics and correlative factors of the hepatic dysfunction. Results Patients who had at least one abnormal result of liver function test accounted for 70.99% in untreated group and 61.07% in treated group. The most common abnormal hepatic function parameter in patients with thyrotoxic hepatic lesion was increased alkaline phosphate enzyme (ALP), which was 57.3% in untreated group and 51.0% in treated group. Abnormalities of alanine transaminase (ALT), aspartate transaminase(AST), gamma-glutamyl tyanspeptidase (GGT) and total bilirubin (TBIL) were 28.2% (37/131), 18.3% (24/131), 10.7% (14/131) and 21.4% (28/131) in untreated group and 21.5%(32/149), 15.4%(23/148), 4.7% (7/149) and 20.1% (30/149) in treated group, respectively. The possibility of thyrotoxic hepatic dysfunction was correlated with the patients' age and FT3 and FT4 levels, but not with the genesis and course of disease, sex, family history of hyperthyroidism, or levels of TGAb and TMAb. Conclusion Abnormal results of liver function test are commom in patients with hyperthyroidism. Increased alkaline phosphate enzyme and alanine transaminase are the most common abnormal hepatic function parameters; the possibility of thyrotoxic hepatic dysfunction is correlated with patients' age and the level of thyroid hormones.
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Objective To construct an animal model of Graves' disease (GD) by immunizing BALB/c mice with hM12 cells co-expressing major histocompatibility complex (MHC) class Ⅱ molecules and human thyrotropin receptor (TSHR) molecules. Methods BALB/c mice in experimental group (H-2d) were immunized with hM12 cells Intraper-itoncally every 2 weeks for six times, while mice in control group were immunized with M12 cells. Five weeks later, the thyroids were histologically examined, and serum samples were tested for thyroid-stimulating antibodies (TSAb) and thyroid hormone levels. Results One BALB/c mouse in experimental group developed Graves'-like disease. Total T4 and T3 levels in this mouse were above the upper limit of normal, TSAb activity was displayed in its serum. The thyroid histologically showed the features of thyroid hyperactivity including thyrocyte hypercellularity and colloid absorption.None of control mice developed Graves'-like disease. Conclusion An animal model with some characteristics of human Graves' disease was successfully induced and the model will facilitate studies aimed directly at understanding the patho-genesis of autoimmunity in Graves' disease.