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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 Arg能够协同
Objective To investigate the role of endogenous nitric oxide (NO) in the induction of apoptosis induced by 5-fluorouracil (5 FU) in Bel74 0 2 hepatocellular carcinoma cells. To explore the mechanism of 5-fluorouracil and to find an adjuvant to improve the clinical efficacy of 5-fluorouracil. Methods Human hepatocellular carcinoma cells Bel74 0 2 were cultured in DMEM medium without L-arginine (L Arg) according to the conventional conditions. Under the action of 5-Fluorouracil, the expression of inducible nitric oxide synthase was detected by immunohistochemistry. Nitrate / nitrite was determined enzymatically with the presence of L arginine, Reflect the generation of NO and prove its function. The morphological changes of apoptotic cells were observed by electron microscope and the apoptotic cells were quantified by flow cytometry. The results were validated by the L-arginine competitive competitor L NAME (Nω nitro L Arginine methylester). The results of immunohistochemistry were quantitatively analyzed by using high-definition pathology color image analysis system. Analysis of variance and t test were used for statistical analysis. Results The expression of inducible nitric oxide synthase was enhanced by 5 fluorouracil (0 16 87 ± 0 0 196 8 in 5 fluorouracil group and 0 10 4 9 ± 0 0 12 6 6 in control group, P 0 05) , And the concentration of endogenous NO was 73 0 ± 10 2 μmol / L (P <0 05) in the presence of its substrate L arginine, which was significantly increased. In human hepatocellular carcinoma Bel74 0 2 cell line The apoptotic rate was significantly higher (17 85 ± 0 78%) and cell necrosis rate was significantly reduced (32 99 ± 0 83%). L NAME antagonized the effect of endogenous NO. Conclusions 5 FU and L Arg are synergistic