Study On Mechanism Of Glucocorticoid Resistant B-Cell Lymphoma Escape Cytotoxicity Of NK Cells

BackgroundLymphoma is a kind of malignant tumour of clonally hyperplastic lymphoid hematopoietic system, B- cell lymphoma takes the big part, The treatment of lymphoma is always multi-drug combination chemotherapy. With more research, the molecular targeted drugs Rituximab comes out, which target to kill antigen with lymphoma cells characteristics and the treatment effect has been obviously improved, but it is still difficult to treat lymphoma which become resestent to multidrugs. In recent years, allogeneic hematopoietic stem cell transplantation and cellular immunotherapy are also used in the treatment of lymphoma, obtaining good results.In allogeneic hematopoietic stem cell transplantation and immunotherapy, NK cell can reduce the incidence of graft-versus-host desase (GVHD) in one hand, and mediate the graft-versus-leukemia (GVL) effect on the other hand, reducing the relapse rate and play and important role in body's anti-tumor immune mechanism. It has said in study that drug resistant tumor cells become less sensitive to NK cell'scytotoxicity effect to escape and survival as the source of tumor recurrence. This study use dexamethasone (DXM) to induse drug-resistance of B-cell lymphoma SU-DHL-4(SU) and build the SU/DXM cell line, observe the difference of cytotoxicity activity of NK cell against SU and SU/DXM cells, and analyse the occurrence molecular mechanisms by RT-PCR, flow cytometry and other methods.ObjectiveThe objective is to study the correlated molecular mechanisms of B-cell lymphoma escapecytotoxicity mediated by NK cell.Research MethodsChapter I The biological characteristics of human lymphoma B-cell SU cells and SU/DXM cells.Added 20μg/ml DXM into RPMI1640 to culture SU cells, until it growed up stably. Observed morphology and growth characteristics of SU and SU/DXM cells under light microscope, flow cytometry was used to analyze cell cycle distribution of SU sell, the counting method was used to draw cell growth curve, using MTT method to detect the IC50 value and resistance spectrum. Chapter II Cytotoxicity activity of NK cells against SU and SU / DXM cell in vitroNormal peripheral blood mononuclear cells were obtained from 3 healthy volunteers, NK cells were obtained through flow cytometry as effector cells, SU and SU/DXM cells cultured as target cells, Cytotoxicity of NK cells against SU and SU/DXM cells were measured by CFSE measure when effect-to-target (E:T) cell ratio was 20:1.ChapterⅢThe correlated molecular mechanisms of cytotoxicity mediated by NK cells against SU/DXM cells.DNA were extracted from NK cells and SU cells; KIR genotype of NK cell and HLAⅠgenotype of SU cells were determined by PCR-SSP. mRNA expressions of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3) in SU and SU/DXM cells were analyzed by RT-PCR. Observe the difference killing activity between NK cell to SU cells and SU/DXM cells when E:T ratio was 20:1. To test whether NK cell killing activity was associated with the expression of target cell surface NKG2D ligands.ResultsChapter I The biological characteristics of human lymphoma B-cell SU cells and SU/DXM cells.1. SU cell is round, grow suspendedly, with large size. G0/G1 cell cycle takes 43.6% of the cellular cycle, S phase cells accounts for 48.5%, G2/M phase cells accounts for 7.97%; dexamethasone-resistant SU/DXM grow suspendedly as well, G0/G1 cell cycle takes 50.9% of the cellular cycle, S phase cells accounts for 35.3%, G2/M phase cells accounts for 13.8%, the G0/G1 part is more than SU cell. there are no obvious different characteristics under the microscope.2. The drug resistance index for SU cells to DXM, ADM, L-asp and Vp-16 are 8.59,3.64,2.98 and 2.61 times than before. The multidrug resistant cell (MDR) is represented as SU / DXM cell.Chapter II Cytotoxicity activity of NK cells against SU and SU/DXM cells in vitro1. NK cells purified by flow filtration could meet the test requirements.2. By CFSE method, It shows that cytotoxicity of NK cells to SU and SU/DXM cells are(25.45±3.21)% and(11.42±2.43)% respectively when ratio of target and effect cells is 20:1 .In the same T/E ratio,cytotoxicity differences between groups of NK cells to SU cells and SU/DXM cells are statistically meaningful (P <0.05).ChapterⅢThe correlated molecular mechanisms of cytotoxicity mediated by NK cells against SU/DXM cells.1. NK cells of all 3 normal cases show KIR genes,including KIR2DL1,KIR2DL3,KIR3DL1 and KIR3DL2.2. Genotypes of SU cells are A 68,68;B 15(B75),15(B75);Cw 03,08。3. SU and SU/DXM cell express various ligand genes of NKG2D in level of mRNA. RT-PCR results shows that, MICA, MICB, ULBP2 gene expression of SU / DXM cell decreased significantly compared with SU cells. The differences have significantly statistical meaning.(P <0.05).Conclusion1. Lymphoma SU cells can become MDR cells with using DXM in a long time.2. Cells occurred MDR could resist not only the drugs'killing, but also cytotoxicity mediated by NK cells.