【摘要】 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更多还原

【Abstract】 Objectives: To evaluate the effects of the endogenus nitric oxide on the apoptosis induced by 5 fluorouracil in liver carcinoma cell line Bel7402 and prove the new mechanism of the antitumor effects of 5 fluorouracil and find effective adjvant for 5 FU. Methods: The cells were cultured under routine conditions using Dulbeccos modified Eagles medium (DMEM) without L Arginine(L Arg). The expression of inducible nitric oxide synthase(iNOS) and apoptosis of the cells induced by 5 fluorouracil with sufficient L Arg added in the medium were observed. The production of nitric oxide was evaluated by determination of the expression of iNOS in the cells and the concentration of nitrite and nitrate in the supernant. The morphology of apoptosis cells was observed by using electronic microscope. The quantity of apoptosis cells was recorded by flow cytometry. The expression of iNOS was detected by immunohistochemical staining and analyzed by utilizing the pathological picture analyzing system. Oneway analysis of variance and t test was applied for statistical analysis. Results: 5 fluorouracil had coordinative effects with L Arg by increasing the production of endogenous nitric oxide. 5 fluorouracil can increase iNOS expression (5 Fu group 0.1687±0.01968; control group 0.1049±0.01266,P< 0.05). The concentration of nitric oxide of the experiment group was 73.0±10.2 μmol/L, significantly higher as comparing to the other two groups(P< 0.05). Thus 5 fluorouracil can induce the production of nitric oxide. The apoptosis rate of the cells was 17.85±0.78%,which was also higher than that of the other two group P< 0.05). The necrotic cell rate and the reminant cell rate were significantly decreased by 32.99±0.83% and 3.18±1.01%,respectively,as compare to 5 fluorouracil group(P< 0.05). Nω nitro L Arginine methyl ester (L NAME),the antagonist of L Arg, can block the changes of those phynomena. Conclusions: 5 fluorouracil can induce the expression of iNOS resulting in increase the production of nitric oxide the medium with enough L Arg. The sensitivity of the cells to 5 fluorouracil was increasd following increased production of nitric oxide. Nitric oxide plays important role in the process of 5 fluorouracil inducing the apoptosis in liver carcinoma cells. L Arg is the most important substrate of iNOS to produce nitric oxide, so it could be a good adjvant of the chemo therapy.更多还原