全反式维甲酸诱导荷瘤裸鼠EC9706食管癌细胞凋亡的机制

Mechanism of apoptosis of esophageal cancer EC9706 in nude mice induced by all-trans retinoic acid

目的 探讨全反式维甲酸（ATRA）诱导荷瘤裸鼠EC9706食管癌细胞凋亡的作用机制.方法 将食管癌EC9706细胞接种于裸鼠,成瘤后将裸鼠分为ATRA作用组、5-氟尿嘧啶（5-Fu）作用组、联合用药组和空白对照组,每组10只,腹腔内连续给药10 d后处死裸鼠.采用原位末端标记（TUNEL）法,检测各组裸鼠移植瘤组织中的细胞凋亡情况.采用免疫组化SP法和逆转录-聚合酶链反应（RT-PCR）,检测caspase-3和survivin蛋白及mRNA的表达水平.结果 ATRA作用组、5-Fu作用组和联合用药组的细胞凋亡率分别为44.3%、39.7%和91.0%,均明显高于空白对照组（0.7%,均P＜0.01）.空白对照组caspase-3蛋白表达水平为46.12±0.33,caspase-3 mRNA的相对表达量为0.14±0.03,均显著低于ATRA作用组、5-Fu作用组和联合用药组（P＜0.05）.空白对照组survivin蛋白表达水平为96.07±0.13,survivin mRNA的相对表达量为0.84±0.04,均显著高于ATRA作用组、5-Fu作用组和联合用药组（均P＜0.05）.ATRA作用组与5-Fu作用组的细胞凋亡率、caspase-3和survivin蛋白及mRNA的表达差异均无统计学意义（均P＞0.05）,但单药组与联合用药组的差异均有统计学意义（均P＜0.05）.结论 ATRA可诱导荷瘤裸鼠EC9706细胞产生凋亡,其作用机制与下调survivin蛋白和mRNA的表达以及上调caspase-3蛋白和mRNA的表达有关.

Objective To investigate the mechanism of apoptosis of EC9706 tumor-bearing nude mice induced by all-trans retinoic acid（ATRA）. Methods Human esophageal carcinoma cell line EC9706 cells were inoculated into nude mice to establish the solid tumor model. The tumor models were divided into the following groups： ATRA group, fluorouracil group, the two-drugs combination group, and with an equal volume fraction of solvent as the control group. The nude mice were sacrificed after 10 days of medication.TUNEL staining was used to detect cell apoptosis. RT-PCR was used to detect the expression level of mRNA and immunohistochemistry was used to detect the expression level of protein of caspase-3 and survivin, the apoptosis-related genes in the the tumor tissue. Results The apoptosis rates of the ATRA group, 5-Fu group and ATRA ＋ 5-Fu group were 44.3%, 39.7% and 91.0%, respectively. There was a significant difference in comparison with the control group（0.7%）, and the ATRA group had no significant difference compared with that of the fiuorouracil group（P 〉 0. 05）, but the apoptosis rate of the two-drugs combination group was significantly higher than that in the two single-drug groups（P 〈 0.05）. The average gray value of caspase-3 protein expressed in the control group was 46.12 ± 0.33 and the relative expression of caspase-3 mRNA was 0. 14 ± 0. 03, both were significantly lower than that in the ATRA group, 5-Fu group and the two-drugs combination group（P 〈 0.05）. The average gray value of survivin protein expressed in the control group was 96. 07 ± 0. 13 and the relative expression of survivin mRNA was 0. 84 ± 0. 04, both were significantly higher than those of other groups（P 〈 0.05）. The ATRA group had no significant difference compared with the fluorouracil group（P 〉 0.05）, but the two-drugs combination group was significantly different compared with the single-drug groups（P 〈 0.05）. Conclusion Apoptosis in the EC9706 tumor cells in nude mice can be induced by ATRA. The mechanism may be related with down-regulation of the level of survivin gene expression and up-regulation of the level of caspase-3 gene expression.