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Objective To select and express a human thyrotrophin receptor antibody (TRAb) Fab fragment from phage antibody library constructed with phage display technology. Methods With immobilized antigen, the reconstructed humanized TRAb Fab library was enriched by five rounds panning (adsorption-elution-amplification). The TSAb Fab and TBAb Fab fragment were selected by coated fusion proteins of hTSHRn and hTSHRc. The positive clones were identified and selected by Phage-ELISA. TRAb positive clones were identified by PCR and double restriction enzyme digestion. The soluble TRAb (TSAb, TBAb) Fab fragments were expressed. TRAb (TSAb, TBAb) Fab fragments were identified by Western blotting assay. The DNA fragment was sequenced from the positive clones. Results Following five rounds of biopanning, TRAb (TSAb,TBAb) Fab phage antibody was screened. The enrichment effect reached to 77 times and 94 times. The soluble TRAb (TSAb,TBAb) Fab antibodies were expressed from positive clones and identified by phage ELISA. Western blotting analysis showed that the phage displaying Fab had significant binding activity with antigens. These sequence analysis showed that all of the heavy chain Fd gene and light chain gene were derived from human immunoglobulin variable region. The light chain variable region of the monoclonal 48 was homologous to the immunoglobulin light chain Vλ homology of 94.4%, and the heavy chain variable region of the monoclonal 48 was homologous to the immunoglobulin heavy Fd chain VH4 homology of 88.9%. The light chain variable region of the monoclonal 56 was homologous to the immunoglobulin light chain Vλ homology of 95.6%, and the heavy chain variable region of the monoclonal 56 was homologous to the immunoglobulin heavy Fd chain VH3 homology of 84.6%. Conclusion We have successfully selected TRAb (TSAb, TBAb) Fab fragment from a human phage display immune antibody library.
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Objective Changes of thyroid stimulating antibody(TSAb) and thyroid stimulating blocking antibody(TSBAb) in the treatment of anti-thyroid drugs(ATDs), and the effect of ATDs combining with levothyrocine(LT4) on TSAb and TSBAb were analyzed. Methods Using recombinant Trxfus. TSHRn protein and Trxfus. TSHRc protein as antigens, and TSH receptor antibody(TRAb)-N(TSAb binding hot spots), TRAb-C(TSBAb binding hot spots)in the serum of thyroid disease patients were measured with ELISA. The changes of TRAb-N, TRAb-C over 36 months in 117 TRAb-N positive Graves′ patients with hyperthyroidism were analyzed retrospectively. In the course of treatment, 41 cases as A group with ATDs and LT4 treatment, 76 cases as B group with only ATDs, The changes of TRAb-N and TRAb-C were observed in the two groups. Results (1)According to the change of TRAb-N, 117 TRAb-N positive Graves′ patients with hyperthyroidism were different. In group Ⅰ, 10 patients continued to have persistently positive TSAb and continued to have hyperthyroidism, remission rate 0%. In group Ⅱ, 17 patients showed complicated TRAb-N changes, 12 of 17 patients got relapse, 5 of 17 patients got remission, remission rate 29.4%. And in group Ⅲ, with TRAb-N dropping gradually, 15 of 89 patients got relapse, 74 of the 89 patients got remission, remission rate 83.1%. Three groups were significantly different with x2 test(P0.05). Conclusion TSAb and TSBAb can be used to document TRAb-function, which is significant for us to predict the changes of thyroid function. During ATDs treatment, the temporary early low-dose application of LT4 did not significantly affect TSAb and TSBAb.
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To explore the long-term effects of L-T4 treatment on physical and intellectual development in neonatal congenital hypothyroidism(CH) patients during adolescence and adulthood.14 out of 15 patients,aged 15-31 years old,including 9 males and 6 females,were diagnosed by neonatal screening for congenital hypothyroidism.(1) By treatment for 10 years until adolescence,return visit ages were 12.6-18.1 years old,the height of patients were normal in 14 cases,weight normal in 8 cases,and overweight in 6 cases.Only the first patient of neonatal screening for congenital hypothyroidism height 154 cm,weight of 43.5 kg,which were below the standard.Bone age by X-ray showed 9 normal,1 case of rapid development,4 cases with left wrist bone age retardation.IQ combined Raven's test(CRT) showed 3 cases excellent,7 cases normal,3 cases borderline,and 2 cases low.(2)Treatment for more than 20 years to adulthood,9 cases of return visits (8 cases were screened out) by 23-31 years of age,with 5 males and 4 females,height and bone age were all normal,normal weight,only 2 cases BMI slightly overweight.As to IQ,good were in 7 cases,mild retardation in 2 cases.7 patients received above average education;they were all employed except one.The employed patients were all capable for their jobs.In summary,screening out of 8 patients,1 case were of mild mental retardation (12.5％);while 7 patients(87.5％),both in physical and intellectual levels were as those of the normal population.
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Objective To explore the value and methods of echocardiographic application in percutaneous left atrial appendage(LAA) closure for stroke prevention in patients with nonvalvular atrial fibrillation.Methods 6 male patients with nonvalvular atrial fibrillation were enrolled for percutaneous LAA closure,the mean age was (68.7 ± 5.6) years old,the mean CHADS2 (congestive heart failure,hypertension,age≥75 years,diabetes mellitus,and prior stroke or transient ischemic attacks) score was 3.2 ± 1.0.Rheumatic valvular diseases were excluded by transthoracic eehocardiography(TTE) before closure procedure.Transesophageal echocardiography(TEE) was performed to guide the punctures of the atrial septum and then monitored the operation all through the closure procedure.Diameter of LAA orifice was measure by TEE to help choosing the closure device.Immediate results of closure and complications were inspected by TEE simultaneously.24 hours,7 days,3 months,6 months and 1 year follow-up were performed using TTE.Results All the 6 patients underwent LAA closure successfully.3 LAmbre(Lifetech Scientific,Shenzhen) devices and 3 Watchman(Boston Scientific,Natick,Massachusetts) devices were implanted respectively in the 6 patients.Mean diameter of the LAA orifice was (22.4 ± 3.3)mm,and mean size of the closure devices was (28.0 ± 2.9) mm.2 mm in width residual flow at the inferior edge of closure device existed in 1 ease.No complication was observed.Post-procedure 24 hours and 7 days post-procedure followup showed optimal results in all cases.Conclusions Implantation of both LAA closure devices can be performed with high success rates in patients with nonvalvula ratrial fibrillation,with high risk for stroke,and who either had contraindication or were not willing to accept oral antieoagulation.Echocardiography plays a core role all through the closure procedure and can make it safer and more efficient.
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Objective To evaluate the inhibitory effect of statins on glucose-stimulated insulin secretion (GSIS) of pancreatic islet in rat and to explore its mechanisms. Methods According to the average volume, freshly isolated or 24-hour cultured pancreatic islets were randomly divided into control group( incubated with Kreb-Ringer bicarbonate buffer), the atorvastatin group( incubated with 100 μ mol/L atorvastatin), the fluvastatin group (incubated with 100 μ mol/L fluvastatin)and the pravastatin group (incubated with 100 μ mol/L pravastatin). Stimulated by 2. 8,5. 5,11.1,16. 7 mmol/L and 25.0 mmol/L glucose respectively, the effect of 100 μ mol/L statins on ATP content and GSIS was compared in the four groups. GSIS was performed by the 37℃ bath incubation method and ATP content was measured by chemiluminescence method. Results Incubated with 100 μ mol/L atorvastatin for 30 minutes, in the presence of 16. 7 mmol/L glucose, the ATP content [(9. 54 ± 1. 64) pmol/islet vs ( 12. 33 ± 1.89) pmol/islet] and GSIS (1.60 ± 0. 21 vs 2. 39 ± 0. 30) were significantly reduced in comparison with the control group (P＜0. 05). Cultured with 100 μmol/L fluvastatin for 24 hours, the ATP content [( 10. 24 ±2.01 )pmol/islet vs (12. 31 ±2. 16) pmol/islet] and GSIS (3. 12 ± 0. 32 vs 4. 17 ±0. 37 ) were all significantly decreased at the higher glucose concentration of 16. 7 mmol/L ( P ＜ 0. 05). Conclusion Atorvastatin and fluvastatin may inhibit GSIS by decreasing ATP content in pancreatic islet and the inhibitory effect is related to the strength of its lipophilicity.
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ObjectiveTo observe the changes in TSH receptor antibody (TRAb) and soluble intercellular adhesion molecule-1 ( sICAM-1 ) levels after treatment of propylthiouracil ( PTU ) and methimazole( MMI ) in patients with Graves' disease (GD). MethodsOne hundred and six cases of clinically diagnosed patients with GD were divided into PTU and MMI groups( each group, n =53 ). The patients in two groups were regularly followed for 36 months. TRAb and sICAM-1 were measured with ELISA method. Results( 1 ) The general data of two groups were not significantly different before treatment( all P＞0. 05 ). ( 2 ) There was no difference in TRAb positive rates between two groups before treatment. The clinical remission rates between PTU and MMI groups showed no difference (83.02％ vs88.68％ ). The cure rate was higher in MMI group than in PTU group( 58.49％ vs 37.74％, P＜ 0. 05 ). (3) There existed significant differences in TRAb levels determined before and 6, 12, 24,30, and 36 months after treatment( all P＜0. 01 ), being gradually decreased with time( F=275.48 ,P＜0.01 ). TRAb levels between two groups were significantly different( F=5.86, P＜0. 05 ). (4) sICAM-1 levels at 36 months after treatment compared with the baseline in both groups were statistically different (P＜0. 01 ), but no difference was found between two groups.ConclusionsBoth PTU and MMI improve the immune status of patients with GD,and the immunosuppressive effect of methimazole is more evident.
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Objective Using recombinant TrxTSHRc protein which contains TSBAb antigen epitope as antigen, an enzyme linked immunosorbent assay (ELISA) was established. The cut-off of positive values of TSH receptor antibody (TRAb) was established. Methods Using recombinant TrxTSHRc protein as antigen, the optimum condition was determined, and the indirect ELISA for detecting TRAb (TRAb-C) in the serum of normal control subjects and thyroid disease patients was established. A total of 1 085 patients and healthy controls were tested for this antibody. Results TRAb-C ELISA mainly detected TSBAb. The A405((-x)±s) value in healthy group was 0. 319±0. 107 with a cut-off value ((-x)±2 s)of 0. 533. The positive rate in thyroid disease patients showed significant deviation, with 66. 01 % in patients with Hashimoto's thyroiditis, 41.68% in newly-diagnosed Graves'disease, 6.98% in simple goiter, 11.1% in subacute thyroiditis, 8.7% in nontoxic thyroid nodular goiter, and 3.2% in thyroid adenoma. Conclusion The TRAb-C ELISA mainly detects TSBAb, which is very valuable for autoimmune thyroid disease, especially for the diagnosis, prognosis and therapy evaluation in Hashimoto' s thyroiditis.
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Objective:To investigate the expression of receptor for advanced glycation end products (RAGE)in the thyroid tissue of diabetes mellitus (DM)rats.Methods:The rat model of DM was established by high-lipid diet and vena caudalis injection of streptozocin.The rats of DM models were then randomly divided into three groups:DM group(n=17),insulin intervention group(n=16)and aminoguanidine intervention group(n=16).Nine normal rats were taken as controls.Twelve weeks after the establishment of DM model,rats were sacrificed and the thyroid tissue was taken to determine RAGE mRNA and the protein expression with the method of RT-PCR and Western Blot.Results:The levels of RAGE mRNA and protein expression were higher in the thyroid tissue of DM rats than those in controls (P＜0.05).The levels of RAGE mRNA and protein expression were lower in the thyroid tissue of insulin intervention group and aminoguanidine intervention group than thor of DM group (P＜0.05).Conclusion:The levels of RAGE mRNA and protein expression were up-regulated in the thyroid tissue of DM rats,which can be restrained by controlling blood glucose and blocking the formation of advanced glycation end products.
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Objective To investigate the thyroid ultrastructural pathological changes of diabetes mellitus (DM) rats as well as the intervention effects of insulin and aminoguanidine. Methods Totally 87 rats were treated with streptozotocin to establish DM animal models and divided into DM group(n=27),insulin intervention group(n=32) and aminoguanidine intervention group(n=28),25 rats were taken as normal controls. Twelve and 20 weeks after the animal model establishment, animals were sacrificed, thyroid tissue was taken and ultrastructure was observed. Results In the thyroid of DM rats, follicular epithelial cells present as applanate shape, microvilli were depleted, rough endoplasmic reticulum dilated to irregular vesicular. None pinocytotic vacuole and casual primary or secondary lysosome were seen. Follicular cavity was dilated, colloid in the cavity had higher electronic-density. Interstitial edema, capillary base lamian was thickened at different stage. Proteo-substance deposition with granulo-shape, cloud shape or homogeneity appeared. The number of thyroid parafollicular cells increased. But endocrine granule in parafollicular cells was few. When compared with DM group, the thyroid tissue injury of insulin intervention group and aminoguanidine intervention group were lessened to different degree. Conclusion The hypofunctional thyroid follicular cells, large quantity of proteo-substance deposition in the interstitium and increased parafollicular cells of DM rats may be related with hyperglycemia toxicity. Insulin and aminoguanidine treatment have some protection effects.
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Objective To establish radioiodine therapy in nonthyroid tumor and to investigate 131Ⅰ treatment effect on xenografted ovarian cancer. Methods Based on previous test, xenografted ovarian cancer nude model were established in nude mice. The effects of radioactive isotope 99m TcO-4 imaging and radioiodine 131Ⅰ treatment on xenografted ovarian cancer in vivo were investigated. Results After transferring human sodium/iodide symporter (hNIS) gene, the xenografted ovarian cancer in nude mice was imaged by isotope 99m TcO-4 Moreover,131Ⅰ exerted inhibitory effect on the proliferative activity. Conclusion After the transfection of hNIS gene, 131Ⅰ has inhibitory effect on proliferative activity of xenografted ovarian cancer.
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Objective To assess the factors which affect the activity of isolated islets,and to construct a steady and effective isolation method of rat pancreatic islets.Methods Pancreatic islets were isolated by collagenase method.The glucose-stimulation of insulin secretion(GSIS) in different conditions was performed by batch incubation method and measured by RIA.Results GSIS was improved by BSA;There was a evident decrease of insulin secretion in cultured islets of 7 days,however,no difference was observed between the freshly isolated islets group and the 1～5days cultured islets groups.Compared to the groups of(5.5 mmol/L) and(25 mmol/L) glucose in the culture medium,(11.1 mmol/L) glucose group stimulated higher insulin release.Conclusion BSA,glucose concentration of RPMI1640 medium and the culture period are related to the activity of isolated islets.
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<p><b>OBJECTIVE</b>To examine serum levels of sICAM-1 from normal controls and patients with thyroid diseases (simple goitre, Graves' disease or Hashimoto's thyroiditis) with (125)I-sICAM-1 RIA established in our lab.</p><p><b>METHODS</b>Using (125)I-sICAM-1 RIA, serum sICAM-1 levels of 400 healthy individuals as the normal group and 1020 patients with simple goitre (SG), Graves' disease (GD) or Hashimoto's thyroiditis (HT) were examined for a comporative chinical study.</p><p><b>RESULTS</b>The serum level of sICAM-1 (x +/- s) in the normal group was 168.43 +/- 36.23 micro g/L. There was no significant difference between the normal and SG groups (P > 0.05), whereas the serum levels of sICAM-1 in autoimmune thyroid diseases (GD or HT) were higher than those in the normal or SG groups (P < 0.05, respectively). After GD patients received one of three medical treatments, their serum sICAM-1 levels decreased (P < 0.05). After GD patients were treated and their thyroid function decreased to normal, their serum sICAM-1 levels were lower than those in relapsed GD patients (P < 0.05).</p><p><b>CONCLUSIONS</b>sICAM-1 RIA can be used to examine autoimmune thyroid diseases. Serum levels of sICAM-1 can be used as a parameter in diagnosing autoimmune thyroid disease and in evaluating the effects of therapy, drug administration or relapse in GD.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Goiter , Blood , Diagnosis , Graves Disease , Blood , Intercellular Adhesion Molecule-1 , Blood , Radioimmunoassay , Thyroiditis, Autoimmune , Blood , DiagnosisABSTRACT
Galectin-3 and CD44v6 expressions in thyroid tumors were assayed by immunohistochemistry. The results suggest that galectin-3 expression is one of reliable markers of carcinoma and high CD44v6 expression is related to lymph node metastasis of thyroid carcinoma.
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Objective To express human thyroid peroxidase (hTPO) epitopes gene and apply its products in clinical assay. Methods hTPO epitopes gene was cloned into expression vector pGEX 4T 3 then transformed into E. coli BL21. Expression of hTPO gene was induced by isopropyl ? D thiogalactoside, expressed product (GST hTPO) was purified by affinity chromatography and their immunoactivity was demonstrated. ELISA technique using GST hTPO as antigen was established for determining TPOAb. Serum TPOAb level was determined, and HLA DR antigen, dendritic cells and lymphocytes in the thyroid gland tissue were observed in these same AITD patients.Results GST hTPO acquired from procaryotic expression had high purity and good immunoactivity. The CVs of the ELISA technique established with GST hTPO were between 5.93%～7.59%. A significantly positive correlation was found between the TPOAb levels determined respectively by ELISA and RIA method. Serum TPOAb level and distribution of HLA DR antigen and dendritic cells showed the same ascending tendency following the aggravation of lymphocyte infiltration. Conclusion Product of genetic engineering, GSH hTPO, can be used to establish a clinical assay for TPOAb. The correlation between the level of serum TPOAb and the detriment of thyroid tissue is demonstrated.