Secretory expression of human thyroid stimulating hormone receptor A subunit in sf9 insect cell system / 西安交通大学学报(医学版)

【Objective】 To construct the secretory expression system of insect cells to express the secretory TSHR A subunit protein in the ovarian cells of Spotoma oryzae (sf9). 【Methods】 A recombinant plasmid containing the target protein was constructed, and then the positive bacmid was screened out by the blue and white spots experiment. The verified bacmid was transfected into SF9 insect cells to obtain recombinant baculovirus. The virus was amplified, and the titer level was detected by virus plaque assay. Finally, Western blotting was used to identify the expression of the recombinant protein and optimize the expression conditions. 【Results】 During the construction of the protein expression system, PCR identification and sequencing results confirmed the correctness of the sequences of the recombinant plasmid and the recombinant bacmid. After the transfection of the bacmid, the signs of virus budding were observed in sf9 cells. The virus was collected and amplified. The titer of P1 generation virus was 2×107 pfu/m according to the plaque assay. The recombinant protein was identified by Western blotting and confirmed to be exogenous into the culture medium. The optimal condition for virus infection and protein expression was 72 h after the infection when the multiplicity of infection (MOI) was 1. 【Conclusion】 We constructed an insect cell expression system secreting TSHR 22-289 (55 ku), and the protein could be successfully glycolyzed. This system provides a preliminary basis for the construction and production of its industrial platform and also provides a useful tool for studies on TSHR protein and prevention of GO in the future.

Difficulties and prospects in the treatment of Graves′ disease / 中华内分泌代谢杂志

Although traditional treatment for Graves′ disease(GD) displays some effects, it is imperative to explore new treatment methods. Based on the pathogenesis of GD, biologic agents developed by consumption of B lymphocytes and acting on thyroid stimulating hormone receptor(TSHR), such as monoclonal antibodies, TSHR antagonists and epitopes, can provide more options for patients with GD, and some new drugs are expected to be put into clinical practice. By restoring the damaged immune system and maintaining normal thyroid function continuously, it can avoid the disadvantages of conventional therapies such as long-term treatment, induction or aggravation of Graves ophthalmopathy, permanent hypothyroidism, and other complications. Preliminary experience suggests that thermotherapy is effective and safe for patients with refractory GD. In addition, the traditional Chinese medicine improves the symptoms and thyroid function of GD patients.The emergence of new therapeutic modalities and techniques will provide new approaches for the future treatment of GD and help clinicians to make the best decision.

The role of TSH-THSR-cAMP-PKA signal pathway in the process of iodine uptake in lactating rat mammary gland / 中华地方病学杂志

Objective:To observe the mRNA and protein expression levels of thyroid stimulating hormone receptor (TSHR), protein kinase A (PKA) and sodium iodine transporter (NIS) in mammary gland tissue of lactating rats with different iodine nutrition levels, and to explore the role of thyroid stimulating hormone (TSH)-THSR-cyclic adenosine monophosphate (cAMP)-PKA signal pathway in the process of mammary iodine uptake during lactation.Methods:Using a group design, according to body weight (80 - 100 g), 110 Wistar female rats were randomly divided into normal iodine (NI) group, severe iodine deficiency (SID) group, moderately iodine deficiency (MID) group, moderately iodine excess (MIE) group and severe iodine excess (SIE) group, with 22 rats in each group. Another 22 Wistar male rats were selected, and the feeding situation was consistent with that of NI group. After 3 months of feeding, 24-hour urine samples of female rats were collected, and the female rats were caged with the male rats (5 ∶ 1). After mating, each female rat was fed separately. At 10 days of childbirth, the lactating rats were sacrificed and thyroid and mammary gland tissues were taken. The urinary iodine was determined by arsenic cerium catalytic spectrophotometry. The morphological changes of thyroid and mammary gland tissues were observed by HE staining. The mRNA expression levels of TSHR, PKA and NIS in thyroid and mammary gland tissues were measured by real-time PCR; the protein expression levels of TSHR, PKA, phosphorylated PKA (p-PKA), and NIS in mammary gland tissue were measured by Western blotting.Results:Compared with NI group (162.59 μg/L), the median urinary iodine of female rats in SID and MID groups (3.16, 6.36 μg/L) was lower, and the median urinary iodine of female rats in MIE and SIE groups (2 356.27, 11 507.29 μg/L) was higher ( P < 0.01). HE staining showed that different levels of iodine uptake had different effects on thyroid follicles: most of the follicles in NI group were uniform round or oval; in MID group, the number of small follicles increased, the epithelial cells were monolayer columnar or cubic, the follicular cavity became smaller, and the glia decreased; the follicles in SID group became smaller, and the epithelial cells were columnar or high columnar, with reduced or absent glia in the follicular cavity; pleomorphic changes were found in thyroid follicles in SIE and MIE groups, with some follicles significantly enlarged and some small follicles hyperplasia. Different levels of iodine intake had different effects on mammary duct: compared with NI group, the connective tissue around the mammary duct in SID and MID groups showed obvious fibrosis, while the fibrosis in MIE and SIE groups was significantly reduced. The results of real-time PCR showed that there were significant differences in the mRNA expression levels of TSHR, PKA and NIS in thyroid tissues of lactating rats with different levels of iodine nutrition ( F = 10.73, 92.37, 115.75, P < 0.01). There were statistically differences in the mRNA expression levels of TSHR, PKA and NIS in mammary gland tissues of lactating rats with different levels of iodine nutrition ( F = 40.25, 39.63, 14.92, P < 0.05). Western blotting results showed that there were significant differences in the protein expression levels of TSHR, PKA, p-PKA and NIS in mammary gland tissues of lactating rats with different levels of iodine nutrition ( F = 4.14, 6.73, 8.48, 4.51, P < 0.05). Among them, the protein expression level of TSHR in MIE and SIE groups was lower than that in NI group ( P < 0.05); the protein expression level of PKA in SID and MID groups was higher than that in NI group ( P < 0.05); the protein expression level of p-PKA in SID group was higher than that in NI group, but that in SIE group was lower than that in NI group ( P < 0.05), the protein expression level of NIS in SID group was higher than that in NI group ( P < 0.05). Conclusions:The mRNA and protein expression levels of TSHR are decreased in mammary gland tissues of lactating rats with high iodine intake, while the mRNA and protein expression levels of PKA and NIS are increased in low iodine intake. TSH-TSHR-cAMP-PKA signal pathway may be involved in the regulation of iodine intake in mammary gland tissue of lactating rats, which may protect itself and its offspring.

Parameters affecting the period for being euthyroid in newly-diagnosed Graves’ disease treated with antithyroid agent

Background: There are limited data about the factors affecting the response time to medical treatment in Graves’ disease (GD) although many studies examined the predictors of the relapse after drug withdrawal. The aim of the current study was to evaluate the time for becoming euthyroid under antithyroid drug (ATD) therapy and the parameters influencing this period in patients diagnosed as GD.Methods: Patients with newly-diagnosed GD and decided to treat with ATD initially between March 2017 and September 2018 were retrieved retrospectively. Sociodemographic features as well as laboratory parameters like thyroid function tests and thyroid-stimulating hormone-receptor antibody (TRab) at the time of diagnosis were recorded.Results: Out of 41 patients, 63.4% (n=26) were female. The mean age was 36.1±11.7 years and 43.9% (n=18) of them were smoking. The time between the initiation of treatment and the duration of becoming euthyroid was 2.4±1.8 months. No significant difference was noted between age, gender, and smoking status and the time to become euthyroid under ATD treatment. This period was significantly positively correlated with levels of free triiodothyronine, free thyroxine, and negatively correlated with thyroid-stimulating hormone. Response to ATD therapy was higher in patients with pre-treatment TRab levels <10 IU/l than TRab ≥10 IU/l (p=0.011).Conclusions: Pretreatment thyroid function tests and TRab levels may be taken into consideration before deciding treatment in patients with newly diagnosed GD. It would be useful to design more comprehensive studies so that this proposal can find a response in clinical practice.

Analysis of thyroid stimulating hormone receptor gene mutation in children with hyperthyroidism / 中华内分泌代谢杂志

Objective To explore the characterization of thyroid stimulating hormone receptor(TSHR) gene mutational spectrum in children with hyperthyroidism from Guangzhou. Methods Ninety children were diagnosed with hyperthyroidism from July 2009 to July 2014 in our institute. Their median age at diagnosis was(7.5± 3.4) years, and there were 28 males and 62 females. Mutational analysis were performed by performing polymerase chain reaction (PCR) and DNA direct sequencing of exon 10 of TSHR gene. TSHR gene mutations from 50 unrelated healthy children were served as controls. The correlation between TSHR gene and hyperthyroidism in children was explored. Results A total of 3 mutations were identified in ninety children who were diagnosed with hyperthyroidism, one synonymous mutations(p.V614V), and two missense mutations( p. R707W and p. D727E). Mutation of p. V614V do not change amino acid and do not influence the structure and function of TSHR, no pathogenicity. p.R707W is a SNP associated with human cancers. The frequency of C allele of the D727E in children with hyperthyroidism was 86.7%, while 55.0% in the controls, significant different between the children with hyperthyroidism and the controls( P＜0. 01). In this study, a very high association between the D727E SNP and hyperthyroidism ( OR=18. 86, P＜0. 01) was found. Conclusion Three different mutations of TSHR gene exon 10 were identified in 90 children with hyperthyroidism, (c.1842A＞G,p.V614V、c.2119C＞T,p.R707W、c.2181G＞C,p.D727E), there were association between p.D727E and hyperthyroidism, nor p. V614V and p. R707W. Finally, p. D727E may be correlated with hyperthyroidism in children.

Laboratory Testing in Thyroid Conditions - Pitfalls and Clinical Utility

Thyroid disorders are common, affecting more than 10% of people in the US, and laboratory tests are integral in the management of these conditions. The repertoire of thyroid tests includes blood tests for thyroid-stimulating hormone (TSH), free thyroxine, free triiodothyronine, thyroglobulin (Tg), thyroglobulin antibodies (Tg-Ab), thyroid peroxidase antibodies (TPO-Ab), TSH receptor antibodies (TRAb), and calcitonin. TSH and free thyroid hormone tests are frequently used to assess the functional status of the thyroid. TPO-Ab and TRAb tests are used to diagnose Hashimoto's thyroiditis and Graves' disease, respectively. Tg and calcitonin are important tumor markers used in the management of differentiated thyroid carcinoma and medullary thyroid carcinoma (MTC), respectively. Procalcitonin may replace calcitonin as a biomarker for MTC. Apart from understanding normal thyroid physiology, it is important to be familiar with the possible pitfalls and caveats in the use of these tests so that they can be interpreted properly and accurately. When results are discordant, clinicians and laboratorians should be mindful of possible assay interferences and/or the effects of concurrent medications. In addition, thyroid function may appear abnormal in the absence of actual thyroid dysfunction during pregnancy and in critical illness. Hence, it is important to consider the clinical context when interpreting results. This review aims to describe the above-mentioned blood tests used in the diagnosis and management of thyroid disorders, as well as the pitfalls in their interpretation. With due knowledge and care, clinicians and laboratorians will be able to fully appreciate the clinical utility of these important laboratory tests.

Stable expression of the human TSH receptor in CHO cells and the functional assessment / 中华内分泌代谢杂志

Objective To establish and assess a CHO (Chinese Hamster Ovary) cell line that stably expressed the human TSH receptor. Methods The gene of human TSH receptor was amplified by PCR from the recombinant plasmid that had been constructed previously,and then the target gene was linked to linearized vector GV208. Afterwards, with the reconstructed vector and helper vectors, the target gene was packaged into the lentivirus. The recombinant lentivirus infected the CHO cells and integrated the gene into the chromatin. The established CHO cell line was isolated and cultured and its function was assessed.Results The result of gene test identified the correct sequence of DNA of the transformant. The expression of mRNA in CHO cells transfected with the human TSHR was obviously higher than the cells in control or blank group. Within the range of certain density,the more bovine TSH was used to stimulate the established CHO,the more cAMP would be produced. When stimulated with serum of patients of Graves' disease, the cell line also significantly produced more cAMP. Conclusion The established CHO cell line transfected with the human TSHR can stably express the human TSH receptor and response to the stimulation of ligand of TSH receptor.

Progress in the correlation between thyroid stimulating hormone receptor gene and hyperthyroidism / 中华实用儿科临床杂志

The cause of hyperthyroidism is still not clear.Thyroid stimulating hormone receptor(TSHR) gene is one of the hot topic genes in the etiology of hyperthyroidism.In this review paper,the progress of correlation between TSHR gene and hyperthyroidism was summarized.Results suggested that TSHR gene germline mutations could cause familial non-autoimmune autosomal dominant hyperthyroidism and persistent sporadic congenital non-autoimmune hyperthyroidism.In addition,TSHR gene mutation may also undermine the stability of the TSHR and then become the autoantigens to make producing TSHR antibodies.Which can stimulate thyroid follicular to secrete excessive thyroid hormone and then cause Graves' disease.However,the relationship between TSHR gene and the pathogenesis of Graves' disease still needs further study.

Expression of thyroid-stimulating hormone receptor in thyroid tissue of lactating rats / 中华地方病学杂志

Objective To observe the expressions of thyroid-stimulating hormone receptor (TSHR) protein and mRNA in thyroid gland of lactating rats.Methods Eighty adult Wistar rats (60 females and 20 males),weighting 210-250 g were selected.The 60 female Wistar rats were divided into 6 groups according to their body weight by means of random number table:normal non-pregnant group,lactating for 5,10,15 and 20 days groups and weaning for 5 days group,10 rats in each group.All rats were fed with conventional fodder and tap water freely,and the rats of lactating groups except the normal non-pregnant group cohabited with male rats (3 ∶ 1).Then all rats were killed on the 5th,10th,15th and 20th day after lactation and on the 5th day after weaning to get thyroid tissues.The expressions of TSHR protein and mRNA were determined by immunohistochemical staining and realtime quantitative PCR.Results TSHR protein was expressed in cytoplasm and membrane of rat thyroid follicular cells.The expression of TSHR protein in thyroid gland was significant different statistically between groups (x2 =11.227,P ＜ 0.05); the staining intensity of rat thyroid tissues in the normal non-pregnant gruop (weak,n =2; moderate,n =5; strong,n =3) was stronger than that of rats lactating for 5 days (weak,n =7; moderate,n =3; x2 =5.895,P ＜ 0.05).But the expression of TSHR protein in thyroid tissues in the normal non-pregnant group was not significantly different statistically compared with the expression of TSHR protein in other groups (lactating for 10,15and 20 days) and weaning for 5 days group (all P ＞ 0.05).The expression of TSHR mRNA in thyroid gland was significantly different statistically between groups (F =2.970,P ＜ 0.05); the expression of TSHR mRNA in lactating for 5 days group (0.74 ± 0.13) was lower than that of the non-pregnant group (1.02 ± 0.24,P ＜ 0.05); and the expression of TSHR mRNA in the normal non-pregnant group was not significantly different statistically compared with those of other groups (lactating for 10,15 and 20 days) and weaning for 5 days group (all P ＞ 0.05).Conclusion TSHR is widely expressed in thyroid gland of lactating rats,but relatively lower in early lactation period.

Comparison of Patient Characteristics in Thyroid-Associated Ophthalmopathy with and without Strabismus

PURPOSE: To determine immunochemical and clinical differences in thyroid-associated ophthalmopathy (TAO) patients with restrictive strabismus and without strabismus. METHODS: A retrospective chart review of 15 TAO patients with strabismus (25 eyes) and 24 TAO patients without strabismus (39 eyes) who presented to the Ophthalmology Clinic between August 2011 and December 2013 was performed. Visual acuity, intraocular pressure (IOP), Hertel exophthalmometry, soft tissue score, and enlargement of extraocular muscles on computed tomography (CT) were obtained and compared in each group. Thyroid related autoantibody (thyroid-stimulating hormone receptor antibody, TRAb; thyroid peroxidase antibody, TPOAb; anti-thyroglobulin antibody, TgAb) titers and positive rates were obtained at the time of diagnosis or before treatment and analyzed. RESULTS: The gender and smoking proportion were not significantly different between the 2 groups. The mean age of TAO patients with strabismus was 52.53 years and of TAO patients without strabismus 40.33 years (p = 0.004). The differences in visual acuity and IOP between the 2 groups were not significant. Hertel exophthalmometry showed less proptotis in the TAO with strabismus group than the TAO without strabismus group (16.84 mm versus 18.67 mm). The soft tissue score was not significantly different. The extraocular muscle enlargement rate of TAO with strabismus was significantly higher than in TAO without strabismus group. In the TAO with strabismus group, TRAb level was higher than in the TAO without strabismus group (p = 0.021). CONCLUSIONS: The TAO with strabismus group was older and had higher positive rate, level of TRAb, and extraocular muscle enlargement rate on CT than the TAO without strabismus group. Furthermore, proptosis was less definite in the TAO with strabismus group.

Investigation on peripheral blood TSHR mRNA for differentiation of benign and malignant thyroid nodules / 中华内分泌代谢杂志

Objective To assess the value of peripheral blood thyroid stimulating hormone receptor(TSHR) mRNA determination in differential diagnosis of benign and malignant thyroid nodules.Methods Fine needle aspiration cytology (FNAC) and (or) postoperative histopathology as the gold standard were carried out,the expression of circulating TSHR mRNA was determined by RT-PCR in 33 patients with benign thyroid nodules,39 patients with thyroid cancer,and 20 normal controls.Results TSHR mRNA signals were not detected in normal controls,the positive rate of TSHR mRNA was higher in the group with malignant nodules than the group of benign nodules (91.2％ vs 48.5％,P＜0.01).TSHR mRNA level in the preoperative malignant group was significantly higher than that in the normal,benign,and postoperative cancer groups (all P ＜ 0.01).Using peripheral blood TSHR mRNA for differentiating benign or malignant of thyroid nodule had a sensitivity,specificity,and accuracy of 91.2％,51.5％ and 71.6％,respectively.The sensitivities of TSHR mRNA,FNAC,and these two methods combined in detecting malignant nodules were 91.3％,86.9％,and 100.0％ respectively,while diagnostic accuracies were respectively 84.0％,80.0％,and 92.0％.TSHR mRNA expression showed no significant relationship with sex,age,size,and number of nodule in these patients (all P ＞ 0.05),but it did exhibit significant difference between benign and malignant nodules(P＜0.01).Conclusion The peripheral blood TSHR mRNA could be used as a molecular marker for thyroid cancer,and it would help enhance the preoperative differentiation of benign and malignant thyroid nodules.

Recent advances in molecular targeted therapy and expression of iodine uptake relative protein in papillary thyroid carcinoma with BRAFV600E mutation / 中华内分泌代谢杂志

Type-B RAF (BRAF) gene is one of the most popular genes of thyroid carcinoma in recent studies,and its mutation has significant relationships with the occurrence,development,treatment,and prognosis of papillary thyroid carcinoma(PTC).The influence of BRAFV600E mutation on the expression of iodine uptake relative proteins in PTC cells and the value of molecular targeted therapy with BRAFV600Einhibitors in the clinical treatment of PTC in the future were summarized in this review.

Expression of thyroid-stimulating hormone receptor in mammary gland tissue of lactating rats / 中国地方病学杂志

Objective To observe the protein and mRNA expression of thyroid-stimulating hormone receptor (TSHR) in mammary gland tissue of lactating rats,and to explore iodine uptake mechanism.Methods Eighty adult Wistar rats (60 female and 20 male),weighting 210-250 g were selected.All female Wistar rats were randomly divided into 6 groups according to their body mass:normal non-pregnant group,lactating for 5-,10-,15-and 20-day groups and weaning for 5 days group,10 rats in each group.All rats were fed with conventional fodder and tap water freely.In addition to the normal non-pregnant group,other five groups of female and male rats were mated at 3 ∶ 1,respectively.Then the rats in all groups were killed on the 5th,10th,15th and 20th day after lactation and on the 5th day after weaning to get the mammary gland tissue.The protein and mRNA expression of TSHR were determined by immunohistochemical staining and real-time quantitative PCR.Results TSHR protein was expressed in mammary acinar and ductal epithelial cytoplasm.The expression of TSHR in mammary gland showed significant differences between groups (x2 =14.612,P ＜ 0.05),the staining intensity of mammary gland tissue in normal non-pregnant rats(weak,n =4; moderate,n =6) was weaker than that of lactating for 5 days(weak,n =2; moderate,n =3; strong,n =5) and 10 days groups(barely detectable,n =1;moderate,n =4; strong,n =5; x2 =4.113,5.250,all P＜ 0.05).The expression of TSHR mRNA in mammary gland showed significant differences between groups(F=20.488,P ＜ 0.05); the expression of TSHR mRNA in lactating for 10 days group(0.31 ± 0.06) was higher than that of lactating for 5 days group(0.22 ± 0.04,P ＜ 0.01),and the expression of lactating for 15 days group (0.16 ± 0.08) was significantly lower than that of lactating for 5 days group (P ＜ 0.05).Conclusions TSHR is widely expressed in mammary gland of lactating rats.The iodine uptake of mammary gland is enhanced in early lactation period when the body may be more susceptible to iodine deficiency,therefore iodine should be supplemented reasonably.

Association Between Thyroid Associated Ophthalmopath and Thyroid Autoantibody

PURPOSE: The same autoimmune process is thought to cause thyroid associated ophthalmopathy (TAO) and Graves' disease. The aim of this study is to determine hether thyroid autoantibody is related to the development of thyroid associated ophthalmopathy. METHODS: A retrospective chart analysis was performed on patients with a newly diagnosed Graves' disease, who presented to our ophthalmology clinic between January 2006 and December 2009. Thyroid autoantibody titers were obtained at the time of diagnosis and were used to determine the presence or absence of TAO. In addition, any correlations between thyroid autoantibodies were analyzed in patients with TAO. RESULTS: Thyroid autoantibody levels correlated with the development of TAO. Fifty-eight (69%) out of 84 patients with positive thyroid-stimulating hormone receptor antibody (TRAB) levels at the time of diagnosis had TAO. Only 50 (51%) of the 99 patients with negative TRAB levels had TAO. This difference between the two groups was statistically significant (odds ratio, OR=2.2, p=0.013). A statistically significant correlation with the development of TAO was also found in thyroid peroxidase antibody (TPOAb) and anti-thyroglobulin antibody (TgAb), respectively (OR=0.5, p=0.317; OR=0.3, p=<0.001). In patients with TAO, the correlation between TPOAb and TgAb levels was very high (r=0.64, p=<0.001). CONCLUSIONS: A significant association was determined to exist between the development of TAO and thyroid autoantibody level. This result demonstrates the clinical utility of thyroid autoantibody for the diagnosis of TAO in patients with newly diagnosed Graves' disease.

Risk of non-medullary thyroid cancer influenced by polymorphic variation in the thyroglobulin gene / 中国癌症杂志

Background and purpose: Germline variation in Tg (thyroglobulin) and TSHR (thyroid stimulating hormone receptor) confers an increased risk of benign thyroid disorders. Benign thyroid disorders are strong risk factors for non-medullary thyroid cancer (NMTC). To explore the hypothesis that polymorphic variation in these genes affects the risk of NMTC. Methods: Tg A7589G and TSHR C253A polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (FCR-RFLP) method, to analyze the relationship between the Tg and TSHR gene polymorphisms and NMTC in NMTC and control groups. Results: Among 360 cases, there was no statistic difference in the frequencies of genotype and allele of TSHR C253A between NMTC and control groups. There were Tg A7589G polymorphisms in the 360 cases. The frequencies ofAG+GG genotype in NMTC group were significantly higher than those in control groups (P<0.05). The frequencies of G allele in NMTC group were significantly higher than those in control groups (P<0.001). Conclusion: There were Tg A7589G gene polymorphisms in NMTC and control groups. G allele may be the predisposing gene of NMTC.

Methylation of TSHR Gene Promoter in Papillary Thyroid Carcinoma / 医学研究杂志

Objective To investigate the relationship between the characteristics of promoter methylation of thyroid stimulating hormone receptor(TSHR) gene in papillary thyroid Carcinomas(PTC) and the clinical manifestation of PTC. Methods The methylation status of TSHR gene was detected by methylation specific PCR technique(MSP).Results (1) The methylation rate of TSHR gene in PTC tissues was 64.7%(22/34),while the methylation rate of TSHR gene in adjacent thyroid tissues(ATT) was 26.5%(9/34),and the rate of methylation of TSHR promoter in PTC was significantly higher than of ATT(P

Thyroid-stimulating hormone receptor and its cleavage and shedding / 上海第二医科大学学报

Thyroid-stimulating hormone receptor(TSHR)is an important autoantigen whose cleavage and shedding can induce autoimmunity.The TSHR is the main functional receptor of the thyroid.The shedding of the ? subunit plays an important role in the forming of TSAb and balancing between TSAb and TBAb.Research on the cleavage and shedding of the TSHR extracellualar domain helps to gain a deep insight into the structure and function of TSHR,illuminate the pathogenesis of autoimmune thyroid disease and find out a new therapy.The structure of TSHR,the mechanism and the significance of its cleavage and shedding are reviewed in this article.

Poly-peptide immunogenicity of human thyroid-stimulating hormone receptor that may cause Graves' disease / 基础医学与临床

DQA1*0501.TSHR peptides of 183～198、195～210、248～263、301～320、343～362 and 357～376 could bind with high affinity to both HLA-DR3 and HLA-DQA1*0501,all of their IC50 values less than(1 ?mol/L),but they could bind to neither HLA-DR7 nor HLA-DQA1*0201 with high affinity.Conclusion Epitopes of TSHR 183～198,195～210,248～263,301～320,343～362 and 357～376 on the TSHR extracellular domain might be the auto-antigens to cause GD.

Gene cloning and expression of human TSHR ectodomain in eukaryotic cells / 西安交通大学学报(医学版)

Objective To clone and construct the plasmid containing human thyroid stimulating hormone receptor(TSHR) gene ectodomain,and then identify the immunoreactivity of the purified recombinant protein.Methods TSHR total RNA was extracted from human thyroid,and cDNA was obtained with RT-PCR technique.Human TSHR ectodomain gene(hETSHR) was cloned into pcDNA3.1(+) vector.The recombined construct was transfected into CHO cells by Lipofectin 2000.The transcript mRNA was detected by RT-PCR,and protein immunoreactivity was assayed by TSHR antibody with immunocytochemistry staining and Western blot.Results DNA sequencing results showed that the recombinant of human thyrotropin receptor ectodomain had confirmed sequence reported in GenBank.The fusion protein had the immunoreactivity.Conclusion Human thyroid stimulating hormone receptor was successfully cloned in eukaryotic expression vector and the constructor was expressed in eukaryotic cells very well.