阿司匹林预防小鼠结肠癌肝转移的作用及其机制

Effects and mechanisms of aspirin on liver metastases of colorectal cancer

目的 探讨阿司匹林对结肠癌肝转移模型小鼠结肠癌肝转移的预防作用及其可能作用机制.方法 将20只BALB/c小鼠采用随机数字表法分成对照组和实验组,每组10只.对照组给予生理盐水灌胃0.2 mL/d,实验组给予阿司匹林灌胃30 μg/（g·d）.两组小鼠灌胃60 d后使用结肠癌C26细胞采用脾脏种植且切除脾脏的方法建立小鼠结肠癌肝转移模型.模型建立成功后,实验组和对照组小鼠分别予以阿司匹林和生理盐水灌胃直到处死小鼠.建模后观察小鼠肝转移及生存情况.将结肠癌细胞系C26分为两组,对照组不作处理,实验组用10 mmoL/L阿司匹林处理24 h,分别对两组细胞进行划痕实验和Transwell实验,观察阿司匹林对C26细胞侵袭转移的影响;另外通过RT-PCR和Western blot检测两组细胞上皮间质转化（EMT）相关基因的表达.组间比较采用Student-t检验方法.Kaplan-Meier法绘制生存曲线,生存分析采用Log-rank检验.结果 对照组和实验组小鼠肝内转移瘤数目分别为（4.8±1.9）个和（2.6±1.6）个,肝脏荷瘤质量分别为（504±107） mg和（362±67） mg,两组比较,差异均有统计学意义（t=2.840,3.584,P＜0.05）.实验组小鼠的1个月生存率为80％,与对照组的40％比较明显提高,差异有统计学意义（χ^2=4.418,P＜0.05）.病理检查结果显示：实验组小鼠肝脏肿瘤细胞的数量和残存肿瘤细胞的异型性与对照组比较均减少.划痕实验结果显示：划痕24 h后,对照组C26细胞发生了明显的迁移,而与对照组比较,实验组未见细胞发生移动.Transwell法检测结果显示：对照组和实验组C26细胞穿膜细胞数分别为（253 ±21）个和（148±13）个,两组比较,差异有统计学意义（t=5.101,P＜0.05）.RT-PCR法检测结果显示：对照组和实验组C26细胞中EMT相关基因E-cadherin mRNA相对表达量分别为0.002±0.001和0.005±0.001;波形蛋白分别为1.005±0.286和0.270±0.168,两组比较,差异均有统计学意义（t=-4.606,4.942,P＜0.05）.Western blot法检测结果显示：对照组和实验组小鼠肝脏组织C26细胞中EMT相关基因E-cadherin蛋白相对表达量分别为0.473±0.179和1.585 ±0.410;波形蛋白分别为0.787±0.118和0.280 ±0.133,两组比较,差异均有统计学意义（t=-5.542,6.355,P＜0.05）.结论 阿司匹林干预结肠癌肝转移模型小鼠,可抑制结肠癌细胞在肝内的种植转移,提高小鼠生存率.其可能作用机制是阿司匹林上调E-cadherin的表达水平,下调波形蛋白的表达水平,抑制了结肠癌细胞EMT的发生,从而减弱了肿瘤细胞的侵袭和转移.

Objective To investigate the preventive effects of aspirin on liver metastases of colorectal cancer in mice and study the mechanisms.Methods Twenty BALB/c mice were divided into the control group and the experimental group according to the random number table with 10 mice in each group.Mice in the control group were fed with saline each day at a concentration of 0.2 mL/d for 60 days,while mice in the aspirin group were fed with aspirin each day at a concentration of 30 μg/（g · d） for 60 days.Then C26 colon cancer cells were injected into the spleen and then the spleen was cut to establish mice model of colon cancer liver metastasis.The C26 colon cancer cells were divided into 2 groups.C26 colon cancer cells in the control group remained untreated,and C26 colon cancer cells in the experimental group were treated with aspirin at a concentration of 10 mmol/L for 24 hours.The scratches and transwell assays were conducted to observe the effects of aspirin on the invasion and metastasis of C26 colon cancer cells.The expressions of epithelial-mesenchymal transition （EMT）-related genes were detected using RT-PCR and Western blot.All data were analyzed using the Student t test.The survival curve was drawn by Kaplan-Meier method,and the survival analysis was done by Log-rank test.Results The numbers and weights of hepatic metastatic tumors were 4.8 ± 1.9 and （504 ± 107） mg in the control group and 2.6 ± 1.6 and （362 ± 67） mg in the experimental group,with significant difference between the 2 groups （t =2.840,3.584,P 〈 0.05）.The 1-month survival rate was 80% in the experimental group,which was significantly higher than 40% of the control group （χ^2=4.418,P 〈 0.05）.The results of pathological examination showed that tumor cell heteromorphism was reduced by aspirin.The results of scratches experiment showed an obvious migration of C26 colon cancer cells in the control group at 24 hours later,while no C26 colon cancer cells migrated in the experimental group.The numbers of C26 colon cancer cells penetrated the Watrige were 253 ± 21 in the control group and 148 ± 13 in the experimental group,with significant difference between the 2 groups （t =5.101,P 〈0.05）.The relative mRNA expression of the E-cadherin and the Vimentin were 0.002 ±0.001 and 1.005 ±0.286 in the control group and 0.005 ± 0.001 and 0.270 ± 0.168 in the experimental group,with significant difference between the 2 groups （t =-4.606,4.942,P 〈 0.05）.The relative protein expressions of the E-cadherin and the Vimentin were 0.473 ±0.179 and 0.787 ± 0.118 in the control group and 1.585 ± 0.410 and 0.280 ± 0.133 in the experimental group,with significant difference between the 2 groups （t =-5.542,6.355,P 〈 0.05）.Conclusion Aspirin inhibits liver metastasis of colon cancer and promote the survival ratio of mice.Aspirin can up-regulate the expression of E-cadherin and down-regulate the expression of Vimentin,which inhibits EMT and reduces the invasion and metastasis of tumor cells.