| Objective: Malignant lymphoma is one of the most dangerous malignant tumors in childhood with an inceasing incidence. The pathogenesis and disease mechanism of lymphoma are required for being demonstrated more clearly. New strategies for treatment are urgently needed. Since it was known the EDFR was nitric oxide (NO), many researches about NO were made. NO is catalysed by nitric oxide synthase with L-arginine. In resent researches people found NO plays an important role in tumor occurrence, invasion and metastasis. Possible mechanisms of NO's promoting tumors are its capacity to stimulate angiogensis and to inhibit apoptosis. In-situ expression of iNOS and synthesis of NO have been show in several malignant tumors including colon, breast, and lung cancer. Little has been elucidated on the potential role of iNOS and NO to the paediatric malignant lymphoma and lymphoma cells. In this study to elucidate the role and the possible mechanism of iNOS and NO in the paediatric malignant lymphoma, we studied the expression of iNOS and the relationship between iNOS and VEGF and clinical characters. At the same time iNOS mRNA-positive human Burkitt's lymphoma BJAB cells were used to investigate the effects of NO on lymphoma cells. Methods: The study comprised 31 cases of malignant lymphoma and 8 cases of normal controls, all of which were under 18 years. All patients were all hospitalized in the Fourth Affiliated Hospital of Hebei Medical University and the Hebei Children's Hospital between Dec, 1990 to Dec, 2004. The normal controls were hopspitalized at the paediatric surgery of the Second Affiliated hospital of the Hebei Medical University considered their lymph nodes amputated in the operations as normal ones expect inflammation and malignant tumor patients. The diagnostic criterion was based on the seventh editon of Textbook of Paediatrics. The different stage of HD was baesd on An Arbor stage system, NHL based on St.Jude. All biopsied specimens were fixed 10% buffered formalin and embedded in paraffin wax. The expressions iNOS and VEGF were measured by immunohistochemistry including 31lymphoma and 8 normal lymph nodes. The tumor MVD was valued by CD34 monoclonal antibody marker. Burkitt's lymphoma BJAB cells cultured in vitro were divided into two groups, the control group and the test group. The control group had the same Hank's concentration to the test group. The test group was treated respectively with the NOS substrate L-arginine and specific NOS inhibitors NG-monomethyl-L-arginine (L-NMMA) at concentrations of 0.01mM, 0.1mM, 1mM, 5mM. All the cells were incubated for72 hours. Proliferations of BJAB cells were evaluated by MTT assay. Nitrite, the stable end product of NO in solution, was measured using a colorimetric assay based on the Griess reaction to evaluate the amount of NO produced. iNOS was detected by the chemistry way. Results: 1 The expression iNOS was negative in the controls and positive 74.19% in the lymphoma patients. There was significant difference between the two group by exact probabilities (P<0.05). The expression of VEGF was negative in the controls and positive 80.65% in the lymphoma patients. The difference was significant (P<0.05). 2 In the patients, the relation between the expression of iNOS and VEGF was positive correlation (r=0.531, P<0.05). In the patients the MVDs of iNOS-positive and iNOS-negitive were 34.77±21.21, 20.85±10.38 respectively, which was significantly different by independent-sample t test (P<0.05). 3 In the patients, there were 25 NHL, who were further analyzed. No significant difference existed in sex, pathological types and clinical stages by exact probabilities (P>0.05). 4 MTT assay demonstrated that incubation with L-arginine for 72 hours could accelerate BJAB cells growth. The percentage of proliferation in control cells was regarded as 100%. After incubated with L-arginine at the concentrations of 0.01mM, 0.1mM, 1mM, 5mM. The percentages of proliferations in BJAB cells were 106.88±6.13%, 119.23±14.49%, 125.54±14.53%, 125.69±3.31% respectively. Compared with the control group the percentages of proliferations were significant by one-way ANOVA except the group of 0.01mM concentration (F=9.952, P<0.05). On the contrary, incubation with L-NMMA could result in growth inhibition. After incubated with L-NMMA, the percentages of proliferations decreased to 98.07±7.15%,87.02±20.88%,77.12 ±9.57% ,69.85 ±8.90% respectively. At the concentrations of 1mM and 5mM, there was significant difference compared to the control group (P<0.05). 5 After incubated for 72 hours, the concentration of nitrite was 668.54±72.35μmol/L in the control group. After treated with L-arginine at the concentrations of 0.01mM,0.1mM,1mM,5mM, the concentrations of nitrite were 674.16±37.17μmol/L, 764.04 ±10.34μmol/L, 789.14 ±6.19μmol/L, 792.13 ±45.00μmol/L respectively. It was siginificantly higher than that of the control at the concentrations of 1mM group and 5mM group (P<0.05). On the other hand, after treatment with L-NMMA at different concentrations, the concentrations of nitrite in the supernatante of BJAB cells were 662.55 ±19.20μmol/L, 637.83±33.71μmol/L, 558.05±39.95μmol/L, 543.45±133.58μmol/L respectively. At the concentration of 5mM group significant difference existed compared to the control group (P<0.05). 6 In the similar, after incubated for 72hours, the concentration of iNOS was 1.77±0.25 U/ml in the control group. After incubated with L-arginine at different concentrations, the concentrations of iNOS were 2.51±0.45 U/ml, 2.53±0.20 U/ml, 2.83±0.80 U/ml, 3.48±0.68 U/ml respectively, which was siginificantly lower than that of the control (P<0.05) except the group of 0.01mM concentration (P>0.05). While after incubation with L-NMMA, the concentrations of iNOS were reduced to 2.10±0.79 U/ml, 1.33±0.71 U/ml, 1.11±0.14 U/ml, 0.65±0.09 U/ml as the concentrations of L-NMMA increasing. It was significantly lower than that of the control group at the concentrations of 1mM and 5mM (P<0.05). Conclusions: 1 Paediatric lymphoma could express iNOS and VEGF. The normal lymph nodes were negative. In the lymphoma patients, the expressions of iNOS and VEGF were positive correlation and MVD iNOS-positive was significantly higher than that in iNOS-negative. The results showed that iNOS might involve in vascularization, invasion, deterioration of lymphoma. 2 The expression of iNOS was not related to sex, clinical stages and pathological types in NHL. So it could not be concluded that there was an association between iNOS and clinical characters in NHL.3 Substrate L-arginine could increase iNOS activity and production of NO and accelerate the growth of BJAB cells. NOS inhibitor L-NMMA could decrease iNOS...
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