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Objective The aims of the study were to investigate the effects of human islet amyloid polypeptide (hIAPP) on autophagy in INS-1 cells and its underlying mechanism,and to explore the role of autophagy in hIAPP-induced cytotoxicity and oxidative stress.Methods INS-1 cells were treated with hIAPP (10 μmol/L) for 24 h in the presence or absence of N-acetyl-L-cysteine (NAC),compound C,5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and 3-methyladenine (3-MA),respectively.Transmission electron microscopy was used to observe the number of autophagosome in cells.Cell viability was determined by methyl thiazolyl tetrazolium (MTT) test.2',7'-dichlorofluorescin diacetate (DCFH-DA) assay was used to measure the relative levels of reactive oxygen species (ROS).Western blot was used to detect expression of adenosine monophosphate-activated protein kinase (AMPK) and autophagic markers p62 and microtubule associated protein 1 light chain3 (LC3).Results Treatment of INS-1 cells with hIAPP resulted in a significant increase in the number of autophagosomes and the expression of LC3-Ⅱ/LC3-Ⅰ (both P＜0.05).Meanwhile,treatment of INS-1 cells with hIAPP enhanced the level of ROS to 1.76 times of control cells (P＜0.01).Co-treatment with NAC,an antioxidant,inhibited hIAPP-induced ROS generation,and the expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK in the INS-1 cells (all P＜0.05).Pretreatment of INS-1 cells with AMPK inhibitor compound C suppressed hIAPP and AICAR,an activator of AMPK,induced expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK (all P＜0.05).Autophagic inhibitor 3-MA and compound C aggravated the hIAPP-induced cell death and ROS generation in INS-1 cells (All P＜0.05).The cytotoxic effects of hIAPP were significantly attenuated by co-treatment with AICAR (P＜0.05).Conclusion Autophagy may act as an adaptive mechanism to alleviate hIAPP-induced oxidative damage and toxicity in INS-1 cells.
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Objective This study was conducted to analyze the clinical characteristics and pituitary function of patients with primary empty sella (PES).Methods The clinical data from 123 hospitalized adult patients with PES from January 2010 to May 2016 were retrospectively studied.Results (1) The average age of the 123 (male 43,female 80) PES patients was (59.2 ± 13.6) years (ranging 24-92 years),among whom 61％ patients were in the age group between 50-69 years.(2) The symptoms of the patients included fatigue (56.1％),headache (34.1％),nausea and vomiting (17.9％),gonadal dysfunction (17.1％),visual disturbance (5.7％) and hypopituitarism crisis (3.3％).(3) Hypopituitarism was found in 66 of the 123 patients.Among them,36.6％,31.7％ and 17.1％ were central hypoadrenalism,hypogonadism,and hypothyroidism,respectively.The percentage of hypopituitarism in complete PES was significantly higher than that in partial PES (P ＜ 0.05).(4) Sixteen patients were concomitant with other autoimmune diseases including 11 patients with Graves' disease and 2 with Cushing's syndrome due to adrenal adenoma.Conclusions The incidence of hypopituitarism in PES was 53.7％,in which the pituitary-adrenal axis hypofunction was more common.An overall evaluation of the pituitary function was essential for the patients who had headache and fatigue,or with suspected PES.The patients with hypopituitarism should be given hormone replacement therapy in time and followed up afterword.
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Pituitary hyperplasia may be found in patients with primary hypothyroidism as the decreased thyroid hormone level attenuates negative feedback effect.Sometimes the enlarged pituitary may be misdiagnosed as a pituitary tumor.Patients with long term untreated hypothyroidism often have extremely high level of serum creatine kinase and thus may be misdiagnosed as suffering from myositis.In order to increase the awareness of the nonspecific symptoms of primary hypothyroidism,this article introduces the diagnosis and treatment of a patient with primary hypothyroidism with raised serum creatine kinase level and pituitary hyperplasia.
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In patient with primary biliary cirrhosis,the metabolism of calcium and vitamin D could be affected and osteomalacia and secondary hyperparathyroidism might occurr.Besides,hypoalbuminemia may mask the real level of serum calcium and thus lead to misdiagnosis of coexisting parathyroid adenoma.Therefore,a rare case of parathyroid adenoma associated with primary biliary cirrhosis was herewith presented to call attention to the effect of hypoalbuminemia on serum calcium.
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Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.
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Objective To observe the effect of amylin on the islet β-cells voltage-gated L-calcium channels in rats. Method Patch clamp technique was employed in the observation of the features and changes of electric current of islet β-cells voltage-gated L-calcium channels before and after using amylin. Results In the glucose environment of 5. 5 mmoL/L, the electric current of rat islet β-cells voltage-gated L-calcium channels was activated at-40 mV and reached the peak at about +20 mV, with a peak value of about-120 pA and the insulin secretion level was(0. 76±0. 12) μg/L. Under the stimulation of glucose of 16. 7 mmol/L, the peak current voltage moved to the left and increased up to-140 pA and the level of insulin secretion measured (1.78±0. 13) μg/L. Hatch islet β-cells in amylin at the concentrations of 0. 5, 1.0, 5.0 and 10.0 μmol/L, respectively. It was observed that in the 0. 5 μmol/L and 1.0 μmol/L groups,there was no remarkable change in the peak potential activation voltage, current, and insulin secretion volume in comparison with the control group. However, in the environment of 5.5 mmol/L glucose, the increase of activation voltage of the 5.0 and 10.0 μmol/L groups was-30 mV, with the peak current reduced to approximately-80 pA and-60 pA and the insulin secretion decreased to (0. 49±0. 11) μg/L and (0. 36±0. 12) μg/L respectively. Under the concentration of 16. 7 mmol/L glucose, the activation voltage increased from-40 mV up to-30 mV and the peak current reduced to-80 pA and-40 pA. In the meantime, the insulin secretion decreased respectively to (1.20±0. 13) μg/L and (0. 89±0. 14) μg/L, which is of significance. Conclusion The secretion of insulin is synchronized with the opening of the islet β-cells voltage-gated L-calcium channels at the stimulation of glucose. The amylin inhibition of the insulin secretion is also synchronized with the opening of islet β-cells voltage-gated L-calcium channels and it's in a positive concentration-dependent manner.
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Objective:To construct a human Fab fragment phage display library and provide a platform for human antibody preparation.Methods:Peripheral blood lymphocytes were collected from healthy donor.The heavy chain Fd fragment and light chain of human immunoglobulin's genes were amplified by RT-PCR,and then cloned into phagemid pComb3XSS to generate human phage antibody library.Cutting with endonucleases such as SacⅠ,XbaⅠ,XhoⅠand SpeⅠto identify the insertion of the light chain or heavy chain Fd genes.IL-2 and digoxin as the antigen was used to scan the phage antibody library.Results:A phage antibody library of Fab had 8.4?107 members and it's recombinant rate was 70%.Through DNA sequencing of one positive clone,it was showed that its heavy chain belonged to IgG subvariety and its light chain to ? family.Conclusion:The success of constructing a nave human phage antibody library proves the useful of phage display system in human antibody preparation,and it can be used to select,purify and express of amylin Fab antibody.
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Objective To investigate the relationship between the single nucleotide polymorphism in exon 7(G894T) or the insert/deletion polymorphism of variable number of tandem repeat in intron 4(4b/a) of the endothelial constitutive nitric oxide synthase(ecNOS) gene and the coronary disease in patients with type 2 diabetes.Methods In 80 patients with type 2 diabetes(group DM),92 type 2 diabetic patients with coronary disease(group DH) and 119 healthy controls(group NC),the polymorphism G894T was determined by polymerase chain reaction-restriction fragment length polymorphism analysis, while the polymorphism 4b/a was determined by polymerase chain reaction-variable number of tandem repeat analysis.Results The frequencies of allele T or genotype containing with allele T(GT or TT) in group DH were significantly higher than those in group DM or group NC(all P