Research On The Role And Associated Mechanisms Of Baff And Its Special Receptor BAFF-R In B-NHL Cells And The Clinical Significance Study

Section1:Expression and localization of BAFF and BAFF-R in B-NHLObjective:To study the expression and localization of BAFF and BAFF-R in B-NHL cells.Methods:We used RT-PCR to determine mRNA of BAFF and BAFF-R in B-NHL cell lines Raji, Jeko-1and Pfeiffer, and used Western blot and flow cytometric analyses to determine protein expression of BAFF and BAFF-R in human B-NHL cell lines Raji, Jeko-1and Pfeiffer. Localization of BAFF and BAFF-R protein was determined using immunofluorescence confocal microscopy.Results:RT-PCR analysis showed that BAFF and BAFF-R mRNA is expressed constitutively but in quantitatively variable amounts in all three B-NHL cell lines.Western blot analysis further confirmed that BAFF and BAFF-R protein is expressed in all three cell lines we studied. And BAFF, BAFF-R protein is located on the plasma membrane of both Jeko-1and Pfeiffer cell.Membrane-bound BAFF and BAFF-R protein also can be detected on all three cell lines surfaces by flow cytometry.Conclusion:B-NHL cells express BAFF and its special receptor BAFF-R on the plasma membrane.Section2:BAFF contributes to B-NHL cell survival in vitro and the study of the mechanismsObjective:To study the effects of rhBAFF on B-NHL cells and the mechanisms.Methods:Cell growth was measured by MTT assay in lymphoma cell lines with or without rhBAFF. Western blot was used for detecting the expression of proteins involved in NF-κB, PI3K/AKT, ERK signaling pathways and BCL-2family proteins.Results:BAFF improved B-NHL cells survival.After48h incubation with500ng/ml rhBAFF, the ratio of cell viability of Raji,Jeko-1,Pfeiffer was125.28±10.86%,170.89±15.73%,140.77±7.50%respectively,with a1.25,1.71,1.41fold increase compared with the control. With the dose increase of rhBAFF (0.3.90625、7.8125、15.625、31.25、62.5、125、250、500ng/ml), the cell viability of Jeko-1increased.BAFF triggered NF-κB, PI3K/AKT, ERK signaling pathways in B-NHL cells, as shown by the phosphorylation of the IκBα, AKT, ERK, the augmentation of p52and P65; BAFF triggered BCL-2family by the augmentation of anti-apoptotic protein BCL-2, MCL-1, BCL-XL, and the down regulation of the proapoptotic protein BAXConclusion:BAFF can promote the survival and proliferation of B-NHL cells through regulate the NF-κB, PI3K/AKT, ERK signaling pathways and the BCL-2family members.Section3:Serum BAFF and APRIL levels in B-NHL patients and the clinical significanceObjective:To study the serum BAFF and APRIL levels in B-NHL patients and the relationship between BAFF, APRIL and clinical features. To study the relationship between BAFF and APRIL, IL-10.Methods:77patients pathologically diagnosed with B-NHL and65healthy volunteers at the Second Affiliated Hospital of Zhejiang University School of Medicine were included in this study between2014.1and2014.12. We used ELISA to determine protein levels of BAFF, APRIL and IL-10in serum of B-NHL patients. The comparisons between the mean values were performed by the Mann-Whitney U test; The association between the categorical variables was assessed by Chi-square test; The correlation between BAFF, APRIL and clinical features was established by the non-parametric Spearman correlation analysis.Results:BAFF serum levels could be determined in48new diagnosed and29recurrent and refractory B-NHL patients. Serum BAFF levels at the time of diagnosis in B-NHL patients were significantly elevated than the health control(1739.91±1013.07pg/ml vs969.25±346.30pg/ml, P<0.05).At the same time, serum BAFF was more higher in recurrent and refractory patients than the new diagnosed patients (3526.01±2080.98pg/ml, P<0.05). When the median value of BAFF was used as a cut-off point between two groups of patients (low≤1465.80pg/ml vs high>1465.80pg/ml), BAFF levels of new diagnosed patients tended to be higher in patients with ECOG≥2, IPI≥2and high LDH levels. Spearman correlation analysis revealed significant correlation between Ann Arbor stage(r=0.323, p=0.031), B2MG(r=0.381, p=0.011), ECOG(r=0.390, p=0.008), IPI(i=0.442, p=0.002) and BAFF.IL-10serum levels could be determined in34new diagnosed and25recurrent and refractory B-NHL patients, the median was20.19pg/ml,6.89pg/ml respectively,were significantly increased when compared with the32healthy donors (0.00pg/ml, P<0.05), the Chi-square test and the Spearman correlation analysis revealed a significant correlation with BAFF (P<10.05).APRIL serum levels could be determined in39new diagnosed and20recurrent and refractory B-NHL patients.Serum APRIL levels at the time of diagnosis in B-NHL patients were significantly elevated than the health control (3.44±2.57ng/ml vs1.33±0.86ng/ml,P<0.05).Serum BAFF in recurrent and refractory patients was almost equal to the new diagnosis patients (3.45±2.98ng/mI,P>0.05). At the same time, the Chi-square test and the Spearman correlation analysis failed to reveal significant correlation between the patient age, sex, Ann Arbor stage, B symptoms, extranodal disease, LDH, B2MG, ECOG, IPI, Ki67and APRIL (P>0.05).Conclusion:Serum BAFF levels at the time of diagnosis were significantly associated with ECOG, IPI, LDH, B2MG, Ann Arbor stage and IL-10levels which are markers for B-NHL aggressiveness.