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目的:观察内源性NO在5-氟尿嘧啶(5-fluorouracil,5-FU)诱导人肝癌细胞凋亡中的作用,为进一步研究5-FU的作用机理和提高5-FU的临床疗效寻找辅剂。方法:人肝癌细胞Bel-7402按常规条件培养于含10%小牛血清且无L-精氨酸(L-Arg)的DMEM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/亚硝酸盐量,反映NO的生成,并证明它的作用。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-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-FU的作用下,诱生型一氧化氮合酶表达增强(5-FU组0.16870±0.01968,对照组0.10490±0.01266,P<0.05),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,有显著性提高(P<0.05);人肝癌Bel-7402细胞株的凋亡率明显增高(17.85±0.78)%,细胞坏死率和残渣率减少犤分别为(32.99±0.83)%,(3.18±1.01)%犦,与5-FU组相比P<0.05,有统计学意义。L-NAME能够拮抗内源性NO的作用。结论:内源性NO参与5-FU诱导肝癌细胞凋亡。5-FU通过诱导诱生型一氧
Objective: To observe the role of endogenous NO in the apoptosis of human hepatocellular carcinoma cells induced by 5-fluorouracil (5-FU), and to find an adjuvant for further studying the mechanism of action of 5-FU and improving the clinical efficacy of 5-FU. . METHODS: Human hepatoma cells Bel-7402 were cultured in DMEM medium containing 10% fetal calf serum and no L-arginine (L-Arg) under normal conditions. Under the effect of 5-FU, the expression of inducible nitric oxide synthase was observed by immunohistochemistry, and the amount of nitrate/nitrite was determined by enzymatic method under the premise of its substrate L-Arg. Reflect the generation of NO and prove its effect. The morphology of apoptotic cells was observed by electron microscopy and the apoptotic cells were quantified by flow cytometry. The results of the study were validated with the L-Arg competitive antagonist L-NAME (Nω-nitro-L-Argininemethylester). High-resolution pathological color image analysis system was used to quantitatively analyze the optical density of the immunohistochemical results. Statistical analysis was performed using analysis of variance and t-test. Results: Under the effect of 5-FU, the expression of inducible nitric oxide synthase was increased (0.16870±0.01968 in 5-FU group and 0.10490±0.01266 in control group, P<0.05), in the presence of its substrate L-Arg. The concentration of endogenous NO was (73.0±10.2)μmol/L, with a significant increase (P<0.05); the apoptosis rate of human hepatoma Bel-7402 cell line was significantly higher (17.85±0.78)%, cell necrosis. The rate and rate of residue reduction were (32.99±0.83)% and (3.18±1.01)%, respectively, and P<0.05 compared with 5-FU group, statistically significant. L-NAME antagonizes the effects of endogenous NO. CONCLUSION: Endogenous NO participates in 5-FU-induced apoptosis of hepatoma cells. 5-FU induced by induced oxygen