| Part 1: Construction and characteristics of rituximab-resistant cell lines of human diffuse large B-cell lymphomaObjective Drug resistance and recurrence in patients with diffuse large B-cell lymphoma are the main reasons for treatment failure.In order to search for rituximab-resistant-related molecules and explore mechanisms of rituximab resistance,we induced rituximab-resistant cell lines of human diffuse large B-cell lymphoma.Methods In this part,the stepwise increasing dose of rituximab combined with high-dose intermittent shock was used to establish the rituximab-resistant cell model of human diffuse large B-cell lymphoma(SU-DHL-6-R and NU-DUL-1-R).7AAD flow cytometric staining was used to detect the complement-dependent cytotoxicity of rituximab on rituximab-resistant cells and parental cells;CFSE/7AAD/Annexin V flow cytometric staining was used to detect antibody-dependent cytotoxicity of rituximab on rituximab-resistant cells and parental cells;Cell growth curve of rituximab-resistant cells and parental cells were determined by CCK8 method;and cell cycle and cell surface antigen expression of parental cells and rituximab-resistant cells were determined by flow cytometry;Finally we used second-generation sequencing technology and Illumina Infinium Human Methylation 850 K bead chip to detect human diffuse large B-cell lymphoma cell line(NU-DUL-1)and rituximab-resistant cell line(NU-DUL-1-R)screening the differentially expressed genes and differentially methylated genes.Results Two rituximab-resistant cell models NU-DUL-1-R and SU-DHL-6-R of human diffuse large B-cell lymphoma were established successfully.Compared with the parental cell lines,the complement-dependent cytotoxicity and antibody-dependent cytotoxicity of rituximab on rituximab-resistant cell lines were significantly reduced.According to the cell growth curve,the cell proliferation rate of the two rituximab-resistant cell lines in the logarithmic growth phase were slower compared with the parental lines;Cell cycle analysis by flow cytometry showed that the proliferation index of rituximab-resistant cell lines tended to decrease but without statistically significance when compared with the parental cell lines;CD20 showed lower expression on the two rituximab-resistant cell lines.The expression of CD55,CD59 on SU-DHL-6-R was significantly higher than its parental cells(SU-DHL-6)(P<0.05),while there was no significant difference in the expression of CD55 and CD59 between NU-DUL-1-R and its parental cells(NU-DUL-1).289 differentially expressed genes between NU-DUL-1 and NU-DUL-1-R were successfully screened.Compared with NU-DUL-1,126 differentially expressed genes were up-regulated and 163 differentially expressed genes were down-regulated;11656 differentially methylated Cp G positions and 758 differentially methylated regions were screened out.Combined analysis of genes corresponding to differentially methylated Cp G positions and transcriptome differential genes between NU-DUL-1 and NU-DUL-1-R found that PLD4,MS4A7,FCRL5,PREX1,RASGRP3,ADAM28,RINL,TINAGL1 genes had differences in both transcriptome level and DNA methylation level.Compared with NU-DUL-1,TINAGL1 was hypomethylated and up regulated in NU-DUL-1-R;FCRL5 and RASGRP3 were hypomethylated and down regulated in NU-DUL-1-R;while PLD4,MS4A7,PREX1,RASGRP3,ADAM28,RINL were Hypermethylated and down regulated.Conclusion We successfully established rituximab-resistant cell lines of human diffuse large B-cell lymphoma and analyzed the characteristics of them.The combined analysis of differentially expressed genes and differentially methylated genes found that 8 genes(TINAGL1,PLD4,MS4A7,FCRL5,PREX1,RASGRP3,ADAM28,and RINL)were different in both transcriptome level and DNA methylation level between the NU-DUL-1 and NU-DUL-1-R,which may be related to rituximab resistance.Part 2: Combination of decitabine and rituximab enhances the killing effect on diffuse large B-cell lymphoma cell linesObjective The combination of R-CHOP and small molecule drugs to overcome drug resistance is currently a research hotspot.Preliminary clinical trials found that decitabine may improve the treatment effect of relapsed/refractory diffuse large B-cell lymphoma patients.So we explored whether decitabine increased the sensitivity of diffuse large B-cell lymphoma cell lines to rituximab and preliminarily explored its mechanism in order to provide a new theoretical basis for immunochemotherapy of decitabine combined with anti-CD20 monoclonal antibody.Methods 20% fresh normal human serum was used as the source of complement,and 7AAD flow cytometric staining was used to detect the complement-dependent cytotoxicity of rituximab combined with decitabine on the diffuse large B-cell lymphoma cell lines;CFSE labeled tumor cells and then co-cultured with peripheral blood mononuclear cells,7AAD and PE-Annexin V flow cytometric staining was used to detect the antibody-dependent cytotoxicity of rituximab combined with decitabine on tumor cells.The effect of decitabine on the expression of CD20 on diffuse large B-cell lymphoma cell lines was detected by flow cytometry;the effect of decitabine on Treg and Th17 in the co-culture system of peripheral blood mononuclear cells and tumor cells was detected by flow cytometry and q PCR;Elisa was used to detect the effect of decitabine and rituximab on cytokines in the cell supernatant and flow cytometry was used to detect the changes in the proportion of Th17 cells in the co-culture system of peripheral blood mononuclear cells and tumor cells.Results For complement-dependent cytotoxicity,the combination of decitabine and rituximab group had higher cell mortality than the single-drug group on NU-DUL-1-R,SU-DHL-6 and SU-DHL-6-R(P<0.05).For NU-DUL-1,the combined group had higher cell mortality than the single-drug group when the concentration of decitabine was 20 n M(P<0.05);the combined group had higher cell mortality than the single-drug group when the concentration of decitabine was 5n M,but there was no statistical difference compared with the rituximab group(P>0.05).For antibody-dependent cytotoxicity,the combined group induced higher cell mortality than the single drug on NU-DUL-1,SU-DHL-6 and SU-DHL-6-R.For NU-DUL-1-R,the combination group had higher cell mortality than the single-drug group when the concentration of decitabine was 20 n M(P<0.05);the combination group had higher cell mortality than the single-drug group when the concentration of decitabine was 5n M,but there was no statistical difference compared with the decitabine group(P>0.05).Decitabine increased the proportion of Treg in the co-culture system of peripheral blood mononuclear cells and tumor cells,and decreased the proportion of Th17 detected by flow cytometry and q PCR.We found that the combination of decitabine and rituximab reversed the increasing proportion of Th17 and the up-regulation of IL17 caused by rituximab detected by flow cytometry and Elisa.We also found that decitabine promoted the secretion of IFN-γ and TNF-β in the co-culture system of peripheral blood mononuclear cells and tumor cells(P>0.05).Decitabine promoted CD20 expression on diffuse large B-cell lymphoma cell lines.Conclusion The combination of decitabine and rituximab enhanced the killing effect on diffuse large B-cell lymphoma cell lines.The possible mechanism was that decitabine increased the expression of CD20 on diffuse large B-cell lymphoma cell lines and reversed the increasing proportion of Th17 and the up-regulation of IL17 caused by rituximab. |
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