摘要
目的 观察肝癌细胞Bel74 0 2中内源性NO在 5 氟尿嘧啶 (5 FU)诱导其凋亡中的作用 ,为进一步揭示 5 氟尿嘧啶的作用机理 ,同时寻找提高 5 氟尿嘧啶的临床疗效的辅剂。方法 人肝癌细胞Bel74 0 2按常规条件培养于无L 精氨酸 (L Arg)的DMEM培养液中。在 5 氟尿嘧啶作用下 ,采用免疫组化法观察诱导诱生型一氧化氮合酶的表达情况 ,并在其底物L 精氨酸存在的前提下 ,采用酶法测定硝酸盐 /亚硝酸盐 ,反映NO的生成 ,并证明其的作用。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 精氨酸的竞争性拮抗剂L NAME(Nω nitro L Argininemethylester)对研究结果进行验证。应用高清晰度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 氟尿嘧啶的作用下 ,诱生型一氧化氮合酶表达增强 (5 氟尿嘧啶组 0 16 87± 0 0 196 8,对照组 0 10 4 9±0 0 12 6 6 ,P <0 0 5 ) ,在其底物L 精氨酸存在的前提下 ,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细胞株的凋亡率明显增高 (17 85± 0 78% ) ,细胞坏死率显著减少 (32 99± 0 83% ) ,L NAME能够拮抗内源性NO的作用。结论 5 FU和L
Objective To evaluate the effect of endogenous nitric oxide (NO) on the ability of 5 fluourouracil (5 FU) to induce apoptosis in the liver carcinoma Bel7402 cell line, and to observe the anti tumor mechanism and effective adjuvant of 5 FU Methods Cells were cultured under routine conditions with Dulbecco's modified Eagle's medium (DMEM) without L arginine (L Arg) We observed the expression of inducible nitric oxide synthase (iNOS) and apoptosis of cells induced by 5 FU with L Arg added to the medium The production of nitric oxide was determined by the cell expression of iNOS detected by immunohistochemical staining, and by the concentrations of nitrite and nitrate in the supernatant Results 5 fluourouracil significantly increased the iNOS expression to 0 1687±0 01968 ( P <0 05, vs control group), and the concentration of nitric oxide to 213±30 2 μmol/L ( P <0 05, vs control group) The apoptotic cell rate increased significantly to 17 85±0 78%, while the necrotic cell rate decreased to 32 99±0 83% ( P <0 05, compared with the 5 FU group) N ω nitro L Arginine methyl ester (L NAME), the antagonist of L Arg, can block the apoptotic effects of endogenous nitric oxide Conclusions 5 FU had a synergistic effects with L Arg by increasing the production of endogenous nitric oxide Endogenous nitric oxide plays an important role in the process where 5 FU induces apoptosis in liver carcinoma cells L Arg may be a good adjuvant for chemotherapy with 5 FU
基金
TheprojectwassupportedbythegrantsfortheExcellentYouthScientistofShandongProvincialScienceCommission (No 2 0 0 0 3 3 ) .
