ABSTRACT
<p><b>OBJECTIVE</b>To construct the expression vector of siRNA targeting parathyroid hormone 1 receptor (PTH1R) gene and evaluate its effect on the cell cycle of INS-1 cells.</p><p><b>METHODS</b>The sequences of PTH1R gene was retrieved from Genbank, and 4 pairs of oligonucleotides were synthesized and inserted into pSUPERretro RNAi, which was identified by RT-PCR and sequence analysis. The vectors were then transfected into INS-1 cells, in which the expression of PTH1R was observed by Western blotting to evaluate the transfection efficiency. The cell cycle of INS-1 cells in high glucose medium was detected by flow cytometry.</p><p><b>RESULTS</b>RT-PCR and sequence analysis confirmed the correct construction of the siRNA recombinant expression vector targeting PTH1R gene. The vectors were successfully transfected into INS-1 cells, and the most effective vector was selected by Western blotting. Transfection with the siRNA for PTH1R gene silencing resulted in the inhibition of INS-1 form entering the S phase.</p><p><b>CONCLUSION</b>The successful construction of the recombinant PTH1R-siRNA vectors establishes a basis for further study of protective role of the PTH1R gene in INS-1 cells in high glucose medium.</p>
Subject(s)
Humans , Cell Cycle , Genetic Vectors , Genetics , Glucose , Pharmacology , Insulin-Secreting Cells , Cell Biology , Metabolism , RNA, Small Interfering , Genetics , Receptor, Parathyroid Hormone, Type 1 , Genetics , MetabolismABSTRACT
Objective To investigate the expressions of human telomerase reverse transcriptase(hTERT) mRNA and protein in pheochromocytoma and paraganglioma and their significance as diagnostic markers in predicting the biological behaviour of these tumours.Methods Expression of hTERT mRNA was determined by in situ hybridization in 45 pheochromocytomas/paragangliomas(31 benign,7 suspected malignant and 7 malignant) and 9 normal adrenal medulla samples,hTERT protein was determined by immunohistoebemistry.Results hTERT mRNA was expressed in 5/7 malignant turnouts and 5/7 suspected malignant tumours as compared with 3/31 benign tumours(P