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Objective To investigate the effect of serum-deprivation on the changes of [ Ca2+ ] i and the protein release of S100A13 and fibroblast growth factor 1 ( FGF-1 ) in thyroid cancer TT cell,and to reveal the role of Ca2+in the protein release of S100Al3 and FGF-1.Methods The protein expressions of FGF-1 and S100A13 in TT cells under serum-deprivation were detected by Western blot.The released FGF-1 protein from TT cells in the supernatant fluid was detected by ELISA.Realtime dynamic examinations on the change of 1 h [ Ca2+ ] i in TT cells under serum-deprivation were detected by confocal laser scanning microscopy.Then,the effect of EGTA( 2.5 mmol/ L),BAPTA-AM (2.5 μmol/L)on distributions of the fluorescence of S100AI 3 and FGF-1 in TT cells under serumdeprivation for6 h were detected by indirect immunofluorescence.Results The expressions of FGF-1 and S100A13 in TT cells after serum-deprivation for4 h and 6 h were reduced( P＜0.05 or P＜0.01 ),but the released FGF-1 protein from TT cells in the supernatant fluid was elevated ( P＜0.05 or P＜0.01).Confocal laser scanning of Ca2+ imaging indicated that [ Ca2+ ] i of serum-deprivation TT cells maintained the relative stabilization within 23 win,but the rapid rise of [ Ca2+ ] i achieved peak value 1.6 μmol/L after 30 min,and remained stable for about 17 win,and thereafter 40 win slowly dropped to a low level From 40 win to 60 win the [ Ca2+ ] i was about 0.3-0.6 μ mol/L.The average [ Ca2+ ] i was higher than that in normal group,EGTA group,and BAPTA-AM group within 1 h.The protein expressions of S100A13 and FGF-1 did not drop obviously in EGTA group and BAPTA-AM group.Conlusion The release of S100A13 and FGF-1 from TT cell under serum-deprivation is possibly related with the change of [ Ca2+ ]i.Both Ca2+-chelating agents EGTA and BA PTA-AM are able to inhibit the rise of [ Ca2+ ] i and release of S100A 13 and FGF-1 from TT cells under serum-deprivation.
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Objective To investigate whether the release of fibroblast growth factor-1 ( FGF-1 ) was changed after inhibition of S100A13 gene (small hairpin RNA, shRNA)and serum-deprivation in human thyroid cancer cells (TT cells ). Methods The S100A13-shRNA pENTRTM/U6 entry vector was transfected into TT cells. The expression of S100A13 mRNA and protein was detected by immunoflurescence, real-time RT-PCR, and Western blot. Then TT cells were treated with S100A13 gene inhibition and serum-deprivation. The changes in release of FGF-1 were detected by indirect immunoflurescence, RT-PCR, and ELISA. Results S100A13 shRNA transfected TT cells (S100A13 RNAi cells)had a reduction of S100A13 gene and protein expression by 80%.Indirect immunofluorescence indicated FGF-1 was mostly localized in the cytoplasm and nucleus of TT cells in primary culture. When serum-deprivation stress was given to TT cells, FGF-1 in cytoplasm almost disappeared in the cells at 6 h. RT-PCR indicated that when serum-deprivation stress was given to TT cells the mRNA of FGF-1 was reduced. ELISA showed that with inhibition of S100A13, the release of FGF-1 was reduced (P＜0.05).Conclusion S100A13-shRNA pENTRTM/U6 entry vector transfected TT cells may inhibit the expression of S100A13 and reduce the release of FGF-1.
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Objective:To explore the mechanism of islet cell apoptosis caused by glucose at different concentrations.Methods:The islet cells of SD adult rats were prepared by collagenase digestion,purified by Fcoll400 and cultured in RPMI1640.Different concentrations of glucose or mannitol were added into the monolayer of islet cells.The medium insulin concentration was measured by RIA.The apoptosis rate was measured by the flow cytometry and the bax and bcl-2 expressions were detected by immunoblot.Results:①With glucose concentration raised from 11.1 mmol/L to 22.2 mmol/L,the insulin concentration began to raise and reached the peak,then it began to decline.②With glucose at 11.1 mmol/L and 22.2 mmol/L,the apoptosis rate was the highest,while at 5.5 mmol/L,there was no significant difference compared with the controls. For the groups of mannitol-infused,there was no differences of bax or bcl-2 expressions and no difference of the apoptosis rate comparing with the controls.③With glucose infused,the bcl-2 expressions in 11.1 mmol/L and 22.2 mmol/L groups were negative,bax expression was positive;the bax and bcl-2 expressions were negative in the group of 33.3 mmol/L;the bax and bcl-2 expressions in the 5.5 mmol/L group showed no difference compared with the controls.Conclusion:Higher levels of glucose at 11.1mmol/L and 22.2 mmol/L lead to the release of more insulin and the raise of apoptosis rate.But with glucose concentration at 33.3 mmol/L,the release of insulin decreased and the apoptosis rate declined.Hyperosmolality could not cause the apoptosis of the islet cell.
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PURPOSE To investigate the methylation of p53 gene in human thyroid carcinoma. METHODS The DNA of 12 thyroid carcinomas and 5 adjacent tissues of thyroid carcinoma were digested by restriction endonuclease enzymes Hap I and Msp I . Polymerase chain reaction (PCR) followed by agarose gel electrophoresis was used to detect the methylation in the exon 5 of p53 gene. RESULTS 5'-CCGG-3' site was methylated at the exon 5 of p53 gene in 9 thyroid carcinomas. However hypomethylation was found to exist in 5 adjacent tissues of thyroid carcinoma. CONCLUSION Hypermethylation of p53 gene plays an important role in thyroid carcinogenesis and the mutation of p53 gene.