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目的 :观察肝细胞Bel74 0 2自分泌一氧化氮 (NO)在 5 -氟尿嘧啶 (5 FU)诱导人肝癌细胞凋亡过程中的作用 ,进一步揭示 5 FU的作用机理 ,同时寻找提高 5 FU的临床疗效的辅剂。方法 :人肝癌细胞Bel74 0 2按常规条件培养于含 10 %小牛血清的无L 精氨酸 (L Arg)的DMEM (dulbecco’smodifiedeagle’smedium)培养液中。在 5 FU作用下 ,采用免疫组化法观察诱生型一氧化氮合酶 (iNOS)的表达情况 ,并在其底物L Arg存在的前提下 ,采用酶法测定硝酸盐 /亚硝酸盐 ,反映NO的生成 ,并证明其作用。用电镜观察凋亡细胞的形态 ,流式细胞仪进行凋亡细胞的定量。用L Arg的竞争性拮抗剂L NAME(Nω nitro l argininemethylester)对研究结果进行验证。应用病理彩色图文分析系统对免疫组化结果进行光密度定量分析。采用方差分析和t检验进行统计学分析。结果 :在 5 FU的作用下 ,iNOS表达增强 ,5 FU组 (0 1687± 0 0 1968) ,对照组 (0 10 4 9± 0 0 12 66) ,P <0 0 5 ,在其底物L Arg存在的前提下 ,内源性NO的浓度为 (73 0± 10 2 ) μmol/L(P <0 0 5 ) ,有显著性提高 ;人肝癌Bel74 0 2细胞株的凋亡率显著性增高为 (17 85± 0 78) % ,细胞坏死率和残渣率显著减少分别为 (32 99± 0 83) %、(3 18± 1 0 1) %。L NAME
OBJECTIVE: To observe the role of autotransplantation nitric oxide (NO) of hepatocyte Bel74 0 2 in 5-fluorouracil (5 FU) -induced hepatocarcinoma cell apoptosis and to further elucidate the mechanism of 5 FU. Efficacy of the adjuvant. Methods: Human hepatocellular carcinoma cells Bel74 0 2 were cultured in DMEM (dulbecco’s modifiedeagle’s medium) without L-arginine (L Arg) containing 10% calf serum according to the conventional conditions. Under the action of 5 FU, the expression of inducible nitric oxide synthase (iNOS) was observed by immunohistochemistry. Nitrate / nitrite was detected enzymatically in the presence of L Arg, Reflect the formation of NO, and prove its role. The morphological changes of apoptotic cells were observed by electron microscope and the apoptotic cells were quantified by flow cytometry. The results were validated with L Arg, a competitive antagonist of L Arg (Nω nitro l arginine methylester). The pathological color image analysis system was used to quantitatively analyze the optical density of immunohistochemical results. Analysis of variance and t test were used for statistical analysis. Results: The expression of iNOS was enhanced in 5 FU group (0 1687 ± 0 0 1968), control group (0 10 4 9 ± 0 0 12 66), P <0 05, Arg, the concentration of endogenous NO was (73 0 ± 10 2) μmol / L (P <0 05), which was significantly increased. The apoptosis rate of Bel74 0 2 cell line was significantly increased (17 85 ± 0 78)%, the cell necrosis rate and residue rate were significantly reduced (32 99 ± 0 83)% and (3 18 ± 1 0 1)%, respectively. L NAME