内源性一氧化氮在5-FU联合L-Arg治疗裸鼠人肝癌移植瘤中的作用

Effects of endogenous nitric oxide induced by 5-FU and L-Arg on the human liver carcinoma in nude mice

目的:观察内源性一氧化氮(NO)在5-氟尿嘧啶(5-FU)联合L-精氨酸(L-Arg)治疗裸鼠人肝癌移植瘤中的作用。方法:采用BEL-7402细胞株建立裸鼠人肝癌移植瘤模型,分别给裸鼠腹腔注射生理盐水、5-FU和(5-FU+L-Arg),观察各组药物对肿瘤的抑制作用,病理学观察移植瘤的坏死程度和范围,TUNEL法检测细胞凋亡,免疫组化法检测诱导型一氧化氮合酶(inducible nitric oxidesyn-thesase,iNOS)、p16和Bax的表达,化学比色法检测iNOS的活性,硝酸还原酶法检测瘤组织内的NO浓度,对免疫组化结果采用BI2000免疫组化图像分析系统进行光密度测定,统计分析采用SPSS11.0软件进行One-Way ANOVA、Bonferroni、等级资料的秩和检验(Kruskal-Wallis H)及Pearson相关分析。结果:5-FU联合L-Arg能明显抑制移植瘤的生长,移植瘤组织的坏死范围明显增大,肿瘤细胞的凋亡指数增加,移植瘤组织内的iNOS表达与活性明显增加,NO含量增高,NO浓度与Bax和p16的表达有明显相关性。结论:5-FU联合L-Arg对裸鼠人肝癌移植瘤有明显的抑制作用,其机制可能与诱导iNOS表达和活性增强、内源性NO生成增加、NO诱导抑癌基因和凋亡相关基因表达有关。

OBJECTIVE： To study the effects of endogenous nitric oxide induced by 5-FU and L-Arg on the human liver carcinoma model in nude mice. METHODS： The human liver carcinoma model in nude mice was eatablished with BEL-7402 cell and given the normal saline, 5-FU, （5-FU＋L-Arg） by intraperitoneal injection. The tumor size was measured. The necrotic degree and arrange were observed by the microscope. The apoptosis of cancer cell was detected by TUNEL method. The immunohistochemistry method was performed to determine the expression of iNOS, p16 and Bax. The chemical colorimetry was used to test the activity and the nitrate reductase method was adopted to test the concentration of NO in the tumor tissue. BI2000 was used to analyze the immunohistochemistry results. The one-way ANOVA, Bonferroni, Kruskal-Wallis H and Pearson correlated analysis were adopted for the statistical analysis. RESULTS： 5-FU combined with L-Arg inhibited the tumor growth apparently. The necrotic range and apoptosis index were increased. The expression and activity of iNOS were increased in the tumor tissue. The concentration of NO was also increased. The concentration of NO was related to the expressions of p16 and Bax. CONCLUSIONS： 5 FU combined with L-Arg can inhibit the growth of tumor in nude mice. It may be related to inducing the synthesis and increasing the activity of iNOS. The production of NO is increased. NO can raise the expression of apoptosis-related gene and antioncogene.