A Study On NO, NOS, VEGF, IL-6, IFN-γ, Platelet Glycoprotein And RP Levels In Patients With Acute Leukemia And Its Clinical Significance

Objective To find out the changes of nitric oxide (NO),nitric oxide synthase (NOS), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interferon-y (IFN-y), platelet glycoprotein (p-GP), and reticulated platelets (RP) levels in patients with acute leukemias (AL) of different stages, to explore the correlations between NO, NOS and VEGF, IL-6 and IFN-y, RP and reticulated platelet counts (RPC) in AL patients of different stages, we hope to provide some bases for hematological clinic to develop an anti-angiogenesis and cytokines treatment for AL patients.Methods (1) A total of 73 cases of AL patients were selected and NO, NOS concentration of both bone marrow supernatants and serums of them in both incipient and relieved stages were determined by adopting nitric acid reductase method respectively. (2) VEGF concentrations in patient plasma and bone marrow supernatants of incipient and relieved stages were determined using commercially available enzyme-linked immunosorbent assay (ELISA) sandwich kits for human VEGF (JINGMEI BIOTECH CO. Ltd. LOT: 20020415) following the manufacture's instruction. (3) A total of 20 cases of ALL patients were selected and intracellular IL-6, IFN-y expressions of venous whole blood lymphocytes of them at both incipient and relieved stages were screened approximately 10 000 events each by flow cytometry (Coulter ESP ELITE USA). Cells were cultured with ionomycin in 1640 medium andstimulated by PMA for 4 hours. For flow cytometry, FITC-conjugated rat anti-human IFN-y (Cat.No. 554551, Pharmingen), PE-conjugated rat anti-human IL-6 (Cat.No. 554545, Pharmingen), and IgG isotype control (Becton Dicinson Co. Cat. No. 554685, Pharmingen) were used. (4) A total of 83 cases of AL patients were enrolled in this study and venous whole blood of them were collected at both incipient and relieved stages for p-GPs (CD41, CD42, CD61, CD62p) and RP expression detecting by flow cytometry. And RPC was calculated according to the formula RPC=RP X platelet count of whole blood. For flow cytometry, platelet-rich plasma (PRP) was harvested by centrifugation at 1000 rpm for 10 minutes at room temperature (RT). Then FITC- or PE-conjugated rat anti-human antibodies including FITC-IgG control respectively incubated with lOul PRP in 40ul PBS for 20 minutes at RT in the dark. RP were measured by using 50ul PRP and aided to 450ul undiluted thiazole orange (TO) reagent (ReticCount, Becton Dicinson) incubated for 30 minutes at RT in the dark, and acquisition on a flow cytometry (approximately 10 000 events each) was started immediately thereafter.Results (1) Comparisons of bone marrow and serum VEGF, NO and NOS levels of AL patients in different stages: bone marrow NO, NOS and VEGF levels of both incipient and relieved stages of AL cases were remarkably elevated than that of control group, there were statistical difference among them (F test, all PO.001). Compared the incipient stage of AL with those relieved stage of cases, bone marrow NO and VEGF levels decreased obviously (P<0.05, and PO.01 respectively). In terms of serum NO, NOS and VEGF levels, AL of both incipient and relieved stages were higher than that ofcontrol group (F test, all PO.001). Serum NO, NOS and VEGF levels in AL of relieved stages decreased markedly (all P<0.01). (2) Correlations between VEGF and NO, NOS of different samples of AL cases: there was a positive correlation between serum NO and serum VEGF levels in AL cases of different stages (r=0.68, P<0.01). It showed a strong positive correlation between serum VEGF and bone marrow VEGF concentrations in AL of different stages (r=0.87, P<0.01). There were both a positive correlation between serum NO and serum VEGF levels (r=0.40, P<0.01) and a positive correlation between bone marrow NO and bone marrow VEGF levels (r=0.56, PO.01). (3) Comparisons of both bone marrow and serum NO, NOS, and VEGF levels of different AL subsets cases: in ALL cases, both bone marrow and serum levels of NO, NOS, and VEGF of relieved stages were decreased than that of incipient stages (PO.05 and PO.01 r...