Study On The Difference Of Metabolic Characteristics Of Hypotaurine In Glial Cells And Glioma Cells

Background:Glioma,a nervous system tumor with a high mortality rate,is the most common brain tumor and is more common in adults.Nowadays,gliomas are often treated with surgery combined with radiotherapy and chemotherapy,but the survival of patients with gliomas is only extended to about 14 months.Glioma cells are uniquely invasive,while glial cells are normally present in the nervous system.Therefore,studies on the biological behavior of glial cells and glioma cells are of great significance for the treatment of gliomas.Purpose: 1.To understand the growth of human astrocyte cell line 1800-P and standard glioma cell lines U87 MG and U251 MG under cystine stimulation.2.To investigate the expression of 2-aminoethanethiol dioxygenase(ADO)and cysteine dioxygenase(CDO)genes in glial cells and glioma cells under hypoxia conditions;3.To explore the effect of demethylation of glial cells and glioma cells on the expression of CDO gene.Method: 1.The human astrocyte cell line 1800-P and the standard glioma cell lines U87 MG and U251 MG were cultured in EBSS medium containing different concentrations of cystine,and each cell was detected using water-soluble tetrazolium salt(WST).Under the different conditions of the seven-day growth curve,to explore the effect of different cystine concentrations on various cell growth conditions,according to the proliferation of cystine concentration to find the optimal next step.2.After cultured U87 MG,U251MG and 1800-P under the conditions of three groups of cystine concentration in normoxic and hypoxic environment,RNA was extracted and reverse transcribed to DNA.Real-time quantitative PCR(rt-PCR)technique was used to detect the ADO and CDO gene expression in each cell under the conditions using corresponding primers.3.According to the relevant literature and the results in Method 2,U87 MG,U251MG and 1800-P were demethylated,and the CDO gene expression of three cell demethylation and control groups was detected.4.All the experimental data were plotted by using Graghpad prism7.The values were statistically analyzed by Minitab17.0 software using t-test and One-way ANOVA.P <0.05 was considered statistically significant.Result: 1.The growth curves of U87 MG,U251MG,1800-P cells under different concentrations of cystine were successfully obtained,which proved that there was no significant difference in the proliferation of glial cells and glioma cells under different concentrations of cystine,Three groups of cystine concentrations were obtained from the respective proliferation conditions,which were respectively 0 ?M group,10 ?M group and 200 ?M group for the next experiment.2.The U87 MG,U251MG,and 1800-P were cultured in the presence of three concentrations of cystine medium under normoxic and hypoxic conditions,and the glioma cell lines U87 MG and U251 MG were tested for hypoxia using rt-PCR.The expression of ADO gene was higher than that of 1800-P(P<0.05).The expression of CDO gene in 1800-P cells was higher than U87 MG,U251MG(P<0.05),indicating that the expression of ADO gene in glioma cells was significant,mainly by The ADO pathway synthesizes hypotaurine,while the synthesis of hypotaurine in glial cells relies on the CDO pathway.3.According to the data in 2,the ratios of ADO and CDO gene expression in U87 MG,U251MG,and 1800-P cells were calculated.The results showed that ADO expression of U87 MG and U251 MG was significantly higher than that of CDO(ADO/CDO)under different oxygen concentrations and cystine concentrations.>100),indicating that oxygen concentration and cystine concentration have no significant effect on the expression of ADO and CDO genes in glial cells and glioma cells;4.Using rt-PCR technique,the CDO gene expression of three cell demethylation and control groups was detected.The CDO expression in the demethylation group of the standard glioma cell lines U87 MG and U251 MG was significantly higher than that ofthe control group.There was no significant difference in CDO expression between the demethylated group of the 1800-P cells of the cytoplasm and the control group.Conclusion:The hypotaurine can metabolize the biological activity of glial cells,and cystine is the main raw material for the synthesis of hypotaurine.However,ADO and CDO,which are the main pathways for the synthesis of hypotaurine,have different dominance in different cells.In this study,through studies of glial cells and glioma cells,ADO and KDO gene expression under different conditions,it was confirmed that cystine can promote the proliferation of glioma cells in a certain concentration,and glioma cells in normoxic and hypoxic conditions.The ADO gene is highly expressed,however oxygen concentration has no effect on the proliferation and gene expression of glial cells or glioma cells.Moreover,the expression of CDO gene in glial cells was significantly higher than that of glioma cells,and CDO gene expression was activated in glioma cells after demethylation.