| Lymphoma is a malignant tumor of the immune system which originated from lymph nodes and lymphoid tissue.Lymphoma divides into B-cell lymphoma,T cell/NK cell lymphoma and Hodgkin's lymphoma.Lymphoma has high heterogeneity,so the treatment is also very different.The main treatment of lymphoma is still combined chemotherapy in clinical,but the side effects and drug resistance restrict the use of it.Therefore,to develop new effective therapeutic drug and to improve the therapeutic effect of lymphoma has become a research hotspot.With the emergence of anti-CD20 monoclonal antibody,created the first targeted therapy of cancer,a growing number of new treatment targets for B cell lymphoma were found.Ibrutinb,the BCR?B cell receptor?and BTK?Brutons tyrosine kinase?kinase inhibitor,has made breakthrough in the treatment of B cell lymphoma.BCR compounds and their downstream signaling pathways play a crucial role in normal B cells and a wide variety of tumor B cells,regulating the activity of key molecules can selectively promote or inhibit specific B cell expansion.BCR divided into Ig M?Immunoglobulin M?BCR and Ig D?Immunoglobulin D?BCR,and the signaling pathways mediated by m Ig D?Membrane Ig D?involved in BCR signaling pathways.Ig D,including s Ig D?Secreted Ig D?and m Ig D,plays an important role in antigen recognition,Blymphocyte activation and immune response regulation.In clinical,s Ig D increases significantly in Ig D myeloma patients,and s Ig D levels in the blood is also high in patients with infectious diseases and autoimmune diseases,including RA?rheumatoid arthritis?,SLE?systemic lupus erythematosus?,and so on.Our research group has found expression of s Ig D increased obviously in diffuse large B cell lymphoma in preliminary study.Human Ig D protein can increase the expression of IgDR?Ig D receptor?on the surface of Daudi cells,it can also significantly promote the proliferation,reduce the apoptosis and accelerate the transformation process from G1 phase to S phase in Daudi cells.Therefore,the results suggest that Ig D and IgDR may become a new treatment target for B cell malignancies and blocking the interaction between Ig D and IgDR may become a new immune treatment of B cell lymphoma.Our research group built a fusion protein named h Ig D-Fc-Ig?patent applied for?successfully by connecting h Ig D-Fc and h Ig G-Fc together,aimed at blocking Ig D and IgDR pathways specifically.This paper chose Daudi and Ramos cells for in vitro experiments to investigate the expression of IgDR,to compare the affinity of h Ig D?h Ig D-Fc and h Ig D-Fc-Ig combined with IgDR,to detect the role of h Ig D on cell proliferation,apoptosis and cell cycle and the influence of h Ig D-Fc-Ig on the role of h Ig D above-mentioned,to discuss the role of Ig D and IgDR on the pathogenesis of B cell lymphoma,and to provide experimental evidence in support of h Ig D-Fc-Ig as a possible new treatment drug for B cell lymphoma.Objective: To investigate the expression of IgDR on the surface of Daudi and Ramos cells.To compare the affinity of h Ig D?h Ig D-Fc and h Ig D-Fc-Ig combined with IgDR between Daudi and Ramos cells.To detect the role of h Ig D on cell proliferation,apoptosis and cell cycle on Daudi and Ramos cells and the influence of h Ig D-Fc-Ig on the role of h Ig D above-mentioned and possibly mechanism.To discuss the role of Ig D and IgDR on the pathogenesis of B cell lymphoma,and to provide experimental evidence in support of h Ig D-Fc-Ig as a possible new treatment drug for B celllymphoma.Methods: Daudi and Ramos cells were cultured in vitro.Cell proliferation was evaluated by cell counting kit-8?CCK-8?assay.Cell viability was detected by Trypan blue staining assay.The expression of IgDR,the affinity of h Ig D?h Ig D-Fc and h Ig D-Fc-Ig combined with IgDR,as well as cell cycle and apoptosis were measured by FCM.The protein expressions of c-myc,cyclin D3,CDK6,p16INK4 a of Daudi and Ramos cells were measured by Western blot assay.Results: 1.The expression of IgDR on lymphoma cells and the affinity of h Ig D?h Ig D-Fc and h Ig D-Fc-Ig combined with IgDR.The FCM results showed that: Daudi and Ramos cells have the expression of IgDR.Scatchard line of h Ig D-IgDR?h Ig D-Fc-IgDR and h Ig D-Fc-Ig-IgDR binding on Daudi cells were Y=-0.567X+2072.8?r2 = 0.8796?,Y=-3140.2X+107?r2 = 0.6792?,and Y=-901.53X+2×106?r2 = 0.9097?.The KD value was 9.6×10-9M,1.27×10-11 M and 2.21 × 10-11 M.Bmax value was 3656 arbitrary unit/104 cells,3184 arbitrary unit/104 cells and 2219 arbitrary unit/104 cells.Scatchard line of h Ig D-IgDR ?h Ig D-Fc-IgDR and h Ig D-Fc-Ig-IgDR binding on Ramos cells were Y=-0.1583X+194.43?r2=0.6165?,Y=-1146.6X+1011?r2=0.7047?,and Y=-6.7507X+6×108?r 2= 0.8074?.The KD value was 3.4×10-8M,3.48×10-11 M and 2.96×10-9M.Bmax value was 1228 arbitrary unit/104 cells,8.7×107arbitrary unit/104 cells and 8.9×107 arbitrary unit/104 cells.2.The role of h Ig D on cell proliferation,apoptosis and cell cycle on Daudi and Ramos cells and the influence of h Ig D-Fc-Ig on the role of h Ig D above-mentioned.Daudi and Ramos cells in exponential growth were stimulated with differentconcentrations of h Ig D?1,3,10 ?g/ml?at different times?12,24,36,48,72h?.The results showed that,compared with the control group,h Ig D significantly promoted Daudi and Ramos cell proliferation.But Daudi and Ramos cells in exponential growth were stimulated with h Ig D?3?g/ml?and different concentrations of h Ig D-Fc-Ig?0,0.3,1,3,10,30,100?g/ml?at different times?12,24,36,48,72h?.The results showed that,h Ig D-Fc-Ig inhibits the effect of h Ig D on promoting cell proliferation.Annexin-V/PI staining showed h Ig D?3?g/ml?reduced the apoptosis rate of Daudi and Ramos cells,h Ig D-Fc-Ig?10,30?g/ml?recover the apoptosis rate.Cell cycle detection with PI staining showed h Ig D?3?g/ml?decreased the proportion of Daudi and Ramos cells in G1 phase,increased the proportion in S phase and had no significant influence on G2 phase,while h Ig D-Fc-Ig?10,30?g/ml?made Daudi and Ramos cell cycle arrest in G1 phase.We further detected the protein expressions of c-myc,cyclin D3,CDK6 and p16INK4 a of Daudi and Ramos cells.Western blot results showed that compared with the control group,c-myc,cyclin D3,CDK6 expressions in Daudi and Ramos cells were significantly increased,p16INK4 a expression was significantly reduced after h Ig D?3?g/ml?stimulation,but the expression of c-myc,cyclin D3 and CDK6 were down-regulated and p16INK4 a expression was up-regulated after treatment with h Ig D-Fc-Ig?10,30?g/ml?.Conclusions: 1.Daudi and Ramos cells have the IgDR expression.2.All h Ig D,h Ig D-Fc and h Ig D-Fc-Ig can bind to IgDR on Daudi and Ramos cells,and the binding affinity was higher on Daudi than Ramos cells.3.Both on Daudi and Ramos cells,the binding affinity of h Ig D,h Ig D-Fc and h Ig D-Fc-Ig were h Ig D-Fc>h Ig D-Fc-Ig>h Ig D.4.h Ig D accelerates cell cycle progression of Daudi and Ramos cells,reduces apoptosis,promotes cell proliferation,and these functions may be related to the regulation of c-myc and cyclin D3-CDK6-p16INK4 a expressions.5.h Ig D-Fc-Ig blocks the role of h Ig D above-mentioned: inhibiting cell proliferation of Daudi and Ramos cells,inducing apoptosis and arresting cell cycle in G1 phase,and these functions may be involved in blocking Ig D-IgDR pathway. |
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