The Mechanism Of Ouabain-induced Apoptosis In T Lymphoblastic Leukemia Jurkat Cell Lines

Objective:1.In vitro,to explore the anti-tumor mechanism of ouabain by detecting the apoptotic and hTERT and c-myc mRNA and protein expression level of Ouabain-induced T lymphoblastic leukemia Jurkat cell lines.2.The same concentration ouabain affect on primarily normal BM-MNCs.Methods:1.The cultured of human T-cell acute lymphoblastic leukemia(Jurkat cell line)and the isolated and cultured of health adults BM-MNCs.2.The apoptosis induction effect of ouabain on Jurkat cells and health adults BM-MNCs at concentration of 50 nmol/L and 100 nmol/L for 24h and 48h were detected by flow cytometry after Annexin V/PI staining.3.After ouabain treatment Jurkat cells and health adults BM-MNCs,the c-myc and hTERT mRNA expression levels were measured by realtime-PCR.4.After ouabain treatment Jurkat cells and health adults BM-MNCs,the protein expression of hTERT and c-myc were analysed by Western blotting.5.The relative length of the telomeres of ouabain treatment Jurkat cells at concentration of 50 nmol/L and 100 nmol/L for 24h were detected by Flow fluorescence in situ hybridization.Results:1.After effects of different concentrations of different time of ouabain on Jurkat cells,the apoptosis rates of control group?50 nmol/L 24h?100 nmol/L 24h?50 nmol/L 48h?100 nmol/L 48h were 4.23±1.01%,5.67±3.71%,9.63± 4.83%,19.67±4.5%,37.60±11.89%,respectively.the apoptosis rates were significantly increased ouabain treatment(50 nmol/L or 100 nmol/L)in Jurkat cells after 48h(P<0.05).After the same concentration and the same time of ouabain treatments health adults BM-MNCs,.the apoptosis rates of control group?50 nmol/L 24h?100 nmol/L 24h?50 nmol/L 48h?100 nmol/L 48h were 4.13±0.71%?9.13±1.71%?12.63±6.93%?12.90±4.42%?15.23±7.07%,the treated cells had no statistical significance compared with the control group(P>0.05).2.The mRNA level and protein expression of c-myc and hTERT of T lymphocyte leukemia Jurkat cell lines were significantly higher than health adults BM-MNCs.3.After the ouabain concentration(50 nmol/L,100 nmol/L)on the Jurkat cells,the mRNA levels of hTERT and c-myc were significantly decreased(P<0.05).And the reduction of mRNA levels of hTERT and c-myc were positively correlated.After the same concentration and the same time of ouabain treatments health adults BM-MNCs,.the reduction of mRNA levels of c-myc had no obvious significance(P>0.05),the decreased of mRNA levels of hTERT was statistical significance,but not as Jurkat cell lines reduction obviously.4.After the ouabain concentration(50 nmol/L,100 nmol/L)on the Jurkat cells for 24h?48h,the protein expression of hTERT and c-myc were significantly decreased(P<0.05).And the reduction of mRNA levels of hTERT and c-myc were positively correlated.After the same concentration and the same time of ouabain treatments health adults BM-MNCs,.the protein expression of hTERT and c-myc were no statistical significance(P>0.05).5.After ouabain treatment Jurkat cells at concentration of 50 nmol/L and 100 nmol/L for 24h,the relative length of the telomeres shorten.Conclusion:1.Ouabain can induce T lymphocyte leukemia Jurkat cells apoptosis.After the same concentration and the same time of ouabain treatments health adults BM-MNCs,the apoptosis did not change significantly.2.The mRNA level and protein expression of c-myc and hTERT of T lymphocyte leukemia Jurkat cell lines were significantly higher than health adults BM-MNCs,c-myc?hTERT can be used as therapeutic targets of the Jurkat cell lines.3.Ouabain cause the decreased of hTERT,c-myc mRNA and protein expression levels of Jurkat cells,which may be one of the mechanisms of ouabain-induced apoptosis of Jurkat cells.4.After ouabain treatments health adults BM-MNCs,the mRNA and protein expression levels of c-myc had no obvious change,the treated group of hTERT mRNA levels is reduced compared with the control group,but the treated group of hTERT protein expression had no reduce compared with the control group.