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(P<0.01). One of the 117 TRAb-N positive Graves′ patients with hyperthyroidism developed TRAb-C positive hypothyroidism. (2)According to combining with and without LT4 during the treatment of ATDs,the patients were divided into 2 groups(Group A: ATDs combined with LT4; Group B: only ATDs). These 2 groups were significantly different in TRAb-N at baseline and 3 months(P<0.01), TRAb-C between two groups were not significantly different in all times(P>0.05). 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.