| Objective The aims of this study are to establish the human glucocorticoid-resistant diffuse large B lymphoma cell line Toledo/DEX, to observe biological characteristics; and further investigate the mechanisms of glucocorticoid-resistance.Methods The glucocorticoid-resistant human diffuse large B lymphoma cell line was established by being exposed to dexamethasone (DEX), the dose of which was increased gradually and intermittently for long periods of time. Toledo, in the logarithmic growth phage, was incubated for 96 hours.The culture medium contained DEX in the concentration of 1×10-8M at first. Then the cell line was incubated in the culture medium without DEX.Once it re-entered the logarithmic growth phase, the concentration of DEX was improved twice as much as the previous until the concentration of DEX was 1.024×10-5M. Through CCK-8 assay,we detected the differences between the proliferation of the resistant and parental cell line which were exposed in DEX with different time and concentration. We calculated resistance index by detecting the sensitivity of resistant and parental cell line to DEX. At last, we made the resistant cell line separated from the DEX to examine the stabilities of the resistance to dexamethasone.We observed the biological characteristics of resistant and parental cell line. We observed the morphology of the resistant and parental cell line through optical microscope, cell ultrastructure through transmission electron microscopy.We determined the doubling time, the resistance index and growth curve through CCK-8 assay; the immunophenotype and the cell cycle with the flow cytometry; the karyotypes by the means of R-banding; the nodulation of the resistant and parental cell line.We studied in mechanisms of glucocorticoid resistance by detecting the expression of GRa and GRβprotein through western blot. Results Through induced in 6 months, we estabished the glucocorticoid resistant cell line Toledo/DEX and observed the biological characteristics. In morphology, the resistant cell line was the same to the parental cell line.They both growed in suspension. The size of cell lines were heterogenous, round or oval. The volume were larger than lymphocyte.The cytoplasm was few and nucleus was large and heterogenous, which occupied most of the cell. By Wright's staining, the morphology of two types of cell line were no significant different through optical microscope. The morphology of two types of cell line was similar to Burkitt lymphoma through oil immersion. The cell body was large and irregular. The ratio of nuclear to plasma increased. The cytoplasm was light blue,containing the vacuolus. The nucleus was Irregular with the multiple nucleoli and nuclear fragmentation. The morphology of two types of cell line was similar in cell ultrastructure. The resistant cell line had more lamellar processes and microvilli.The immunophenotype of two types of cell line are CD2-, CD3-, CD5-, CD7-,CD10+, CD19+, CD79a+, CD20+, CD38+, CD16-, CD56-, CD22-. The karyotypes were also similar. There were abnormalities of the chromosomes 1,4,7,10. But no clone formed. Cell growth curve showed that the resistance cell line growed slowly. The resistant and parental cell line could not nodulate in nude mice. The resistant cell line had more Gl phase cells, less S phase and G2 phase cells. The resistant cell line was resistant to chemotherapy drug, compared to the parental cell line.The resistance index of dexamethasone, doxorubicin, cyclophosphamide, and vincristine were 1227,2.05,2.63 and 1.86, respectively. The resistance index of DEX and cyclophosphamide had statistical significance. GRa and GR(3 expressed in both cell lines. The expression of GRa downregulated with the times resistant to glucocorticoid. while GR(3 upregulated with the times resistant to glucocorticoid.Conclusion The glucocorticoid-resistant human diffuse large B lymphoma cell line was established by being exposed to dexamethasone (DEX), the dose of which was increased gradually and intermittently for long periods of time.The biological characteristics showed the stability. This study illustrated that the method was feasible. What's more, this study suggest that the abnormalities in the expression of the protein Gra and GRβprotein involved in the mechanisms of glucocorticoid-resistance. |
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