| Objective:B lymphocytestimulator (BlyS) and a proliferation-inducling ligand(APRIL) were recently discovered as the TNF family new members. They have high homology and common receptors (BCMA and TACI). So, they have close relationship. BLyS and APRIL bind to their receptors to influence activation, proliferation and survival of B and T lymphocytes. Through this kind of mechanism, BLyS and APRIL widely participate in immunoregulation, especially closely related to humoral immune dysfunction such as secreting immunoglobulin and generating autoantibodies. Previous study showed abnormal BLyS/APRIL system was related to generation and development of tumor and many autoimmune diseases. At present, study on relationship of BLyS/APRIL system and autoimmune diseases (systemic lupus erythematosus, sjogren syndrome and so on) was more, but study on relationship of BLyS/APRIL system and multiple myeloma(MM) was less. Serum levels of BLyS and APRIL were measured by ELISA. To Investigate function of BLyS and APRIL in multiple myeloma and their correlation with clinical tumor burden prognosis indices. Methods:1 Sample collectionBlood samples were preliminarily divided into control group(n=8) and multiple myeloma group(n=6). Control group was defined as the group of health adults'serum. Multiple myeloma(MM) group was composed of individual serum of three different time points which was the serum of untreated patients (initial treatment group), patients'serum after three courses of treatment (therapeutic metaphase group) and patients'serum after six courses of treatment (therapeutic anaphase group). Finally, serum were divided into four groups icluding control group, initial treatment group, therapeutic metaphase group and therapeutic anaphase group. Using vacuum tubes to extract venous blood of health persons and multiple myeloma patients 4ml respectively. Isolated blood samples were placed 30 minutes at room temperature. Then, blood samples were centrifugated 15 minutes at 2000r/min. Serum was put into different 1. 5 ml centrifuge tubes according to different groups. Samples were stored in -80℃low temperature refrigerator.2 Serum BlyS and APRIL contents by enzyme-linked immunosorbent assay(ELISA)100μl distilled water was added to sample wells. 50μl sample was added to designated wells. Microwell strips were covered and incubated 3 hours at room temperature(18°to 25°C). Microwell strips were emptied and washed 6 times with 400μl Wash Buffer. 100μl of TMB Substrate Solution was added to all wells including blank wells. The microwell strips were incubated for about 10 minutes at room temperature (18°to 25°C). 100μl Stop Solution was added to all wells including blank wells. Microwell reader was blanked and measured colour intensity at 450 nm. Then, serum BlyS and APRIL contents in vaious groups were measured by ELISA.3 The level of serumβ2-MG was assayed by radioimmunoassay(RIA).50μl sample was added to the polystyrene tube. Meanwhile,β2-MG Antiserum 100μl and 125I-β2-MG 100μl was added to it and well mixed. The tube was placed within 30 to 60 minutes. Then, PR separating agent was added to the tube and well mixed again. It was placed for 15 minutes. After 15 minutes, it was centrifugated 15 minutes at 3500rpm. Supernatant was extracted and abandoned. Then, the tube was put into radio-immunity analyzer and the level of serumβ2-MG was assayed in different groups.4 To add up clinical dataLDH, Cr, ALB, Ig, Ca2+, Hb, and percent of plasma in bone marrow came from examination results of health persons and patients in hospital.5 All data were dealt with SPSS 13.0. Continuous variables were presented as mean±s. Data were analyzed through pearson correlation analysis,repeated measurement analysis of variance and the two independent samples t test. The significance was indicated by P value, the statistically significant difference was indicated by P<0.05.Results:1 The serum levels of BLyS and APRIL were significantly higher in initial treatment group than those in therapeutic metaphase group, therapeutic anaphase group and control group.( BLyS: 1.102±0.446 vs 0.595±0.184, 0.355±0.083, 0.641±0.401 ng/ml, APRIL: 1.417±0.391 vs 1.401±0.191, 1.061±0.117, 1.222±0.317 ng/ml, respectively). And the differences were significant (p<0.05). The serum levels of BLyS and APRIL were significantly greater in therapeutic metaphase group compared with therapeutic anaphase group. And there was significant difference in these two groups as well. Between the BlyS level of therapeutic metaphase group and the one of control group had no significant difference (p>0.05). But the APRIL level of therapeutic metaphase group was significantly higher than the one of control group. Meanwhile both of them had significant difference (p<0.05). In therapeutic anaphase group the serum levels of BLyS and APRIL compared with control group which had no significant difference (p>0.05).2 Serum levels ofβ2-MG were significantly higher in initial treatment group than serum levels ofβ2-MG in therapeutic metaphase group, therapeutic anaphase group and control group(β2-MG: 5.114±0.804 vs 1.984±0.409, 2.011±0.518, 2.755±1.469μg/ml, respectively). The differences were significant (p<0.05). There was no significant difference between therapeutic metaphase group and control group (p>0.05). Between therapeutic anaphase group and control group also had no significant difference (p>0.05). There was no significant difference between therapeutic anaphase group and therapeutic metaphase group as well (p>0.05).3 Serum levels of IL-6 was 16.902±6.025 pg/ml.4 Serum levels of LDH were significantly higher in initial treatment group than these in therapeutic metaphase group, therapeutic anaphase group and control group( LDH: 360.071±90.620 vs 189.507±34.893, 150.064±15.385, 150.071±33.253 U/L, respectively). The differences were significant (p<0.01). Serum levels of LDH were significantly greater in therapeutic metaphase group compared with control group. And the difference was significant (p<0.05). Between therapeutic anaphase group and control group also had no significant difference (p>0.05). Serum levels of LDH were significantly higher in therapeutic metaphase group than these in therapeutic anaphase group, the difference was significant (p<0.05).5 There was a positive correlation between the serum levels of BLyS and APRIL in MM group (p<0.05). 5 The serum levels of BLyS and APRIL correlated positively with IL-6,β2-MG, LDH, Ig, Cr, percent of plasma in bone marrow(p<0.05) and correlated negatively with ALB (p<0.05). The level of serum calcium and hemoglobin had no correlation with the serum levels of BlyS (p>0.05).Conclusions:1 With improvement of diease, high expression of BLyS and APRIL in peripheral blood of patients with B-NHL decreased gradually. The result suggested that BLyS and APRIL were related with the incidence of MM2 The serum levels of BLyS and APRIL correlated positively with non-Hodgkin lymphoma prognostic indicators and tumor burden indexes. Therefore, BLyS and APRIL can become new indices that predict MM prognosis. |
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