【作者】 杨娟丽； 金江； 张会芬； 刘翠平； 赵孝先； 朱秀丽； 刁玉巧； 江莲； 戎小平； 曲凡；

【Author】 Yang Juan-Li Jing Jiang Zhang Hui-Fen et al The Fourth Hospital of Hebei medical university & Hebei provincial tumor hospital, Shijiazhuang, 050011, China

【机构】 河北医科大学第四医院暨河北省肿瘤医院疼痛康复科；

【摘要】 目的:探讨诱导型一氧化氮合酶(iNOS)和血管内皮生长因子(VEGF)在小儿恶性淋巴瘤中的表达情况及其与微血管密度(MVD)的关系,观察NO浓度的改变对体外培养的淋巴瘤细胞生长的影响,进而分析NO及iNOS在小儿恶性淋巴瘤发生、发展中所起的作用。为NO制剂参与联合化疗提供实验依据,为小儿恶性淋巴瘤的治疗开辟新途径。方法:1免疫组化染色:收集小儿淋巴瘤患者的淋巴结活检标本31例作实验组。其中HD 3例,NHL 28例。HD中Ⅲ期 1例,Ⅳ期2例;NHL中Ⅲ期11例,Ⅳ期17例;T细胞型 17例,B细胞型11例;男22例,女6例。选取小儿外科就诊住院的非感染非肿瘤患者术中切除的淋巴结8例,作正常对照。所有标本均经免疫组化S—P法染色后检测iNOS和 VEGF表达情况及MVD计数。2、淋巴瘤BJAB细胞体外培养:实验组:培养基中分别加入Hank’s液溶解的底物L- arg和NOS抑制剂NG-单甲基-L-精氨酸(L-NMMA),对照组:培养基中加入与实验组等体积的Hank’s液,终浓度均分别为0．01mM、0．1mM、1mM、5mM。检测细胞增殖率、细胞上清液中亚硝酸盐(NO-2)浓度(间接反映NO的含量) 及上清液中iNOS浓度。结果:1、iNOS在病例组中23例呈阳性,在对照组中均为阴性,差异有显著性(P<0．05)。2、病例组中iNOS表达与VEGF呈明显正相关(r=0．531;P< 0．05);用CD34来标记肿瘤新生血管,发现淋巴瘤标本中 iNOS阳性组MVD值明显高于iNOS阴性组(P<0．05)。3、分析28例NHL组织,未发现iNOS的表达在性别、分期及免疫分型之间有明显差异(P>0．05)。4、淋巴瘤细胞孵育72小时可见随L-arg浓度增加,细胞增殖率逐渐增大,浓度≥ 0．1mM时与对照组比较有统计学意义(P<0．05),加入L- NMMA增殖率逐渐下降,呈剂量依赖性,浓度≥1mM时与对照组比较差异显著(P<0．05)。5、淋巴瘤细胞孵育72小时可见L-arg处理组细胞上清液中NO2-及iNOS浓度均大于对照组,分别当L-arg浓度≥1mM和≥0．1mM时与对照组比较有统计学意义(P<0．05),反之,在L—NMMA处理组, 随L—NMMA浓度增加,NO2-及iNOS浓度逐渐下降,L- NMMA浓度为5mM时NO2-浓度与对照组相比有统计意义 (P<0．05)。浓度为1mM、5mM时iNOS浓度与对照组相比差异显著(P<0．05)。结果:1小儿恶性淋巴瘤组织中iNOS 表达增高,iNOS与恶性淋巴瘤的生长、侵袭有关,起促进作用。2淋巴瘤细胞中iNOS活性呈底物依赖性,合适浓度的NO 可促进淋巴瘤细胞生长,加入NOS抑制剂,NO生成减少,淋巴瘤细胞生长受到抑制。更多还原

【Abstract】 Objective: To study the expression of iNOS and the relationship between iNOS ,VEGF and MVD. 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: 1.The study comprised 31 cases of the paediatric malignant lymphomas and 8 cases of normal controls. The normal controls were hopspitalized at the paediatric surgery considered their lymph nodes amputated in the operations as normal ones expect inflammation and malignant tumor patients. The expressions iNOS and VEGF were measured by immunohistochemistry. The tumor MVD was valued by CD34 monoclonal antibody marker. 2.Lymphoma cells cultured in vitro were divided into two groups, the control group and the test group. The Hank’ s concentration of the control group was the same with 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 0.01 mM, 0.1mM, 1mM, 5mM. All the cells were incubated for 72 hours. Proliferations of lymphoma cells were evaluated. Nitrite, the stable end product of NO in solution, was measured to?evaluate the amount of NO produced. iNOS was detected at the same time. Results: 1 The expression iNOS was negative in the controls and positive 74.19% in the lymphoma patients. There was statistic difference (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 were significantly higher than that of iNOS-negitive (P<0.05). 3 In the patients, there were 28 NHL, who were further analyzed. No significant difference existed in sex, pathological types and clinical stages (P>0.05). 4 MTT assay demonstrated that incubation with L-argi- nine could accelerate BJAB cells growth. Compared with the control group the percentages of proliferations were significant except the group of 0.01mM concentration (F=9. 952, P<0.05). It was dose dependent-on.On the contrary, incubation with L-NMMA could result in growth inhibition. At the concentrations of 1mM and 5mM, there was statistic difference compared to the control group (P <0.05). 5 After BJAB cells treated with L-arginine the concentrations of nitrite were siginificantly higher than of the control at the concentrations of 1mM group and 5mM group (P<0.05). On the other hand, after cells treated with L-NMMA at different concentrations, the concentrations of nitrite in the supernatante were siginificantly lower than that of the control group at the concentration of 5mM group (P<0.05). 6 In the similar, after BJAB cells incubated with L-arginine at different concentrations for 72 hours, the concentrations of iNOS were siginificantly higher than that of the control (P<0.05) except the group of O.OlmM concentration (P>0.05). While after cells incubated with L-NMMA, the concentrations of iNOS were reduced more and more as the concentrati更多还原