Study On Cell Growth Control Of B-cell Non-Hodgkin Lymphoma By B Lymphocyte Stimulator

Objective: B lymphocyte stimulator(BLyS) is a kind of cell growth factor related to immune function. It is able to stimulate B cells proliferation and differentiation specifically and play an important role in humoral immunity. Animal experiment study shows that defective expression or over-expression of BLyS can lead to immune unbalance and induce various diseases. Recently, the research that BLyS is relevant to human immunologic diseases especially to B cell tumor formation and development is still in the starting stage. This kind of research has been becoming a new focus as the diagnosis and treatment of immunologic diseases. In this research, we will adopt MTT colorimetric assay, flow cytometer method and immunohistochemical technique to analyze the effect of BLyS on proliferation, apoptosis and its related pathway of Raji cells (belonging to human lymphoma cell lines) treated with BLyS. To investigate the effect of BLyS on B Lymphoma Cell cell growth control and its mechanism.Methods:1 Raji cells were incubated in culture medium in vitro. ( 1 ) Effect of BLyS that was divided into different concentration group(0.25μg/ml,0.5μg/ml,1μg/ml) and different time group(24h,48h,72h) on the proliferation of Raji cells was measured by MTT colorimetric method. (2)Effect of BLyS that was divided into different concentration group(0.25μg/ml,0.5μg/ml,1μg/ml) and different time group(24h,48h,72h) and anti-IgM on the proliferation of Raji cells was measured by MTT colorimetric method.2 Chemotherapeutic drug(VP-16) induced tumor cell apoptosis. Equal concentration of VP-16 and different concentration(0.25μg/ml,0.5μg/ml , 1μg/ml) of BLyS were added to experimental group. The flow cytometer method was used to measure the effect of BLyS on Raji cells apoptosis induced by VP-16.3 The immunohistochemical technique was used to observe qualitatively that BLyS influenced the expression of NF-ΚB,JNK and P38 in the course of Raji cells apoptosis induced by chemotherapeutic drug.Results:1 MTT assay showed: BLyS promoted Raji cells proliferation obviously. At different time point(24h,48h,72h), different concentration(0.25μg/ml , 0.5μg/ml , 1μg/ml) of BLyS treated Raji cells, compared with control group, OD value of treatment group increased in different degree. And the difference was significant (P<0.05). With the increasing of concentration and effect time of BLyS, OD value increased gradually. In another word, BLyS promoted Raji cells proliferation in dose-dependent and time-dependent manners. The combined action of different concentration(0.25μg/ml,0.5μg/ml , 1μg/ml) of BLyS and anti-IgM(10μl) also promoted Raji cells proliferation. And the difference was significant(P<0.05). At different time point(24h,48h,72h), the group that were treated with BLyS(0.25μg/ml) had no significance(P>0.05) in enhancing B lymphoma cells proliferation compared with another group that were handled by BLyS(0.25μg/ml) and anti-IgM(10μl), but the group that were treated with BLyS(0.50μg/ml, 1.00μg/ml) were higher in enhancing B lymphoma cells proliferation than another group that were handled by BLyS(0.50μg/ml, 1.00μg/ml) and anti-IgM(10μl). And the difference was significant(P<0.05). It was showed BLyS and anti-IgM had synergistic effect on stimulating B lymphoma cells proliferation.2 The result of flow cytometer method showed: Raji cells after 48h treatment with VP-16 appeared typical apoptotic sub G1 peak. Simultaneous addition of both VP-16 and BLyS inhibited apoptosis obviously. The higher concentration of BLyS was, the lower apoptosis rate of Raji cells was. The apoptosis rate was 18.32%,14.56%,7.39% and 3.28% when Raji cells were treated with BLyS at 0, 0.25, 0.5 and 1.0 mg/ml, respectively. The result indicated VP-16 induced Raji cells apoptosis and BLyS inhibited apoptosis induced by VP-16.3 The result of immunocytochemical method showed: staining intensity of NF-κB was weak positive in control group. And NF-κB distributed diffusely in cytoplasmic and nuclear of Raji cells. Compared with control group, the expression of NF-κB had no significance in the group that treated with VP-16. But simultaneous addition of both VP-16 and BLyS increased the number of positive cells and nuclear staining intensity signally. Staining intensity of JNK and P38 was weak positive in control group. JNK and P38 distributed diffusely in cytoplasmic and nuclear of Raji cells. After Raji cells treated with VP-16, nuclear staining intensity enhanced significantly and the number of positive cells increased. Simultaneous addition of both VP-16 and BLyS decreased the number of positive cells and nuclear staining intensity compared with addition of VP-16Conclusions:1 BLyS could promote Raji cells proliferation. BLyS and anti-IgM had synergistic effect on promoting B lymphoma cells proliferation.2 Through enhancing NF-ΚB protein expression and inhibiting JNK and P38 protein activation, BLyS inhibited Raji cells apoptosis induced by chemotherapeutic drug VP-16.3 BLyS inhibited cells apoptosis induced by chemotherapeutic drug relate to enhancing NF-ΚB protein expression and inhibiting JNK and P38 protein activation.