Study On Biological Properties Of L-asparaginase-resistant Acute Lymphoblastic Leukemia Cells

BackgroundThe prognosis of Acute lymphoblastic leukemia has improved over the past decade,in children even more than in adults. However,due to the presence of chemotherapy resistance factors that lead to in many patients are difficult to obtain relief and eventually relapse. Recently, with the cancer stem cell research,the increasing number of studies that show that drug resistance is an important cancer stem cell characteristics,and cancer stem cells are tumor recurrence and metastasis of the initiating cells. Is there exist CSC in the cell of resistant ALL?In this study, we used the methods of real-time quantitative polymerase chain reaction, Flow cytometry, side population cells to study the biological characteristics of L-asparaginase induced Jurkat T lymphoblastic leukemia cells. ALL cell lines resistant to further study the biological characteristics and to understand ALL the mechanism of multidrug resistance and multidrug resistance and the relationship between cancer stem cells,so as to target therapy of leukemia and provide experimental evidence for such reversal.ObjectiveTo study the biological characteristics the resistanced cell of L-asparaginase induced acute T lymphoblastic leukemia.Methods1. In this study,we use the acute T lymphoblastic leukemia cells (Jurkat)as the parental cells,after L-asparaginase incremental resistance induced by Jurkat cells,the establishment of L-asp resistant cell line.Observed morphology and proliferation characteristics,use the method of MTT analysis of drug resistant cell line and resistance index map.2. Collected the cell of Jurkat and Jurkat resistancd to L-asparaginase, using flow cytometry to detect the change of cell surface markers between the two cells.3. Jurkat and Jurkat cells resistant Hoechst33342 staining,compared to verapamil inhibition test,application of flow cytometry to detect the ratio of side population cells.4. With the real-time quantitative polymerase chain reaction analysis the gene expression of ABCG2, Sox2, Oct-4, Nanog between the Jurkat cells and the Jurkat/L-asp cells.Results1. In this study, we successfully established the cells who are suspended growt in the medium there has L-asparaginase at 0.5u/ml stable growth and named resistant cell line Jurkat/L-asp.2. Use the method of MTT to analysis the Resistance spectrum of Jurkat/L-asp, the results show that the resistance index of daunorubicin, etoposide, methotrexate, cytrarabine of Jurkat/L-asp is higher than Jurkat are its resistance patterns of 15.5,9.59,4.28,4.98 times.3. Real-time quantitative polymerase chain reaction amplification showed that the ABCG2 and Sox-2 gene of Jurkat/L-asp cells are higher expressing than Jurkat cells,he difference was significant statistically significant and have a statistically significance (P<0.05). The gene of Oct-4 and Nanog have no express between Jurkat/L-asp cells and Jurkat cells, there are no statistically significance between the two cells(P>0.05).4. There is higher express CD34 and CD13 in the Jurkat/L-asp cells (P<0.05). Logarithmic phase of acute T lymphoblastic leukemia cell line Jurkat with Hoechst33342 staining, compared to verapamil inhibition test, detected by flow cytometry in two cell lines changes in the amount of SP cells, There is exist about 0.4% SP cells in the Jurkat/L-asp cell. The Jurkat cells have no SP cell exist.Conclusions1. The cells of L-asparaginase -resistant are multidrug resistance cells.2. The cells of L-asparaginase -resistant have the characteristics of cancer stem cells.