ABSTRACT
Objective To investigate the mechanism of STAT5-ROS pathway to mediate IM resistance of K562 cells.K562 cells were cultured with imatinib at gradually increased concentrations to generate resistance cell line.Methods CCK-8 assay was used to clarify the resistance ratio.The STAT5A and STAT5B mRNA levels were detected by RT-PCR.Flow cytometry assay was used to detect the level of ROS and cell apoptosis.The expression of STAT5 protein was detected by Western blot.Results Imatinib resistance cell line K562/G was successfully induced by gradually increasing concentrations of IM.The IC50 of K562/G was eighty times higher than K562 by CCK-8.Cell growth curve showed that K562/G was not inhibited in 20 μmol/L imatinib, whereas the K562 cell was significantly inhibited by up to 0.1 μmol/L imatinib.Intracellular level of ROS in K562/G was obviously higher than that of K562 cells(P=0.000 1).The apoptosis ratio of K562 was lower than that of K562/G when the same concentration of IM react on the two cell lines for the same time(P<0.05).The expression of STAT5A and STAT5B mRNA in K562/G was higher than in K562(P=0.000 1,P=0.017 0).Then,the level of STAT5 proteins in K562/G cells was significantly increased (P=0.009 0).Conclusion The STAT5 is highly expressed in CML.STAT5-ROS is closely related to the formation of imatinib resistance of chronic myeloid leukemia.
ABSTRACT
Objective To discuss the clinical application of minimal residual disease in acute myelogenous leukemia(AML) by wt1 mRNA quantitative combined with multi-parameter flow cytometry (FCM).Methods Real time quantitative polymerase chain reaction (qRT-PCR) method was established for detecting wt1 gene expression level in 35 AML patients.The indexes were detected by different subtypes;And 9 cases of ease and 4 cases of recurrence in patients was followed-up and detected the wt1 level.The multiparameter flow cytometry was used to analyze the minimal residual disease in AML.Results The expression of wt1 gene was significantly higher than that of the control group.Significant difference was found(P<0.05).In the newly diagnosed AMLs, wt1 was the highest in M2 and the lowest in M6.Follow-up of 4 AML patients showed that wt1 gene expression level was markedly decreased after CR, but obviously increased after relapse.The proportion of abnormal myeloid cells in different phases significantly changed by FCM.There was no difference of minimal residual disease in AML by qRT-PCR and multiparameter flow cytometry.The ROC curve was used to analyze the recurrent cases to get the threshold value(3.33%).Conclusion The quantitative analysis of wt1 combined with multi-parameter flow cytometry can be used to monitor minimal residual disease in leukemia patients, assess the treatment efficacy and prognosis, and predict the risk of recurrence.