氨氯地平及5-氟尿嘧啶联合用药对小鼠肝癌H22细胞的抑制作用

Inhibitory effect of amlodipine combined with fluorouracil on murine hepatoma H22 cells

目的探讨氨氯地平(amlodipine)及5-氟尿嘧啶(5-fluorouracil,5-Fu)联合用药对小鼠肝癌H22细胞的抑制作用。方法经不同终浓度的氨氯地平(3.125、6.25、12.25、25和50μmol/L)及不同终浓度的5-Fu(12.5、25、50、100和200μg/ml)单独及联合作用小鼠肝癌H22细胞,24、48、72 h后,采用MTT法检测药物对H22细胞生长的抑制作用;流式细胞术及免疫细胞化学法分别检测药物作用48 h后对H22细胞凋亡及细胞中Bcl-2、Bax表达水平的影响。将H22细胞(1.0×107个/ml)经小鼠右腋皮下注射,0.2 ml/只,于注射第2天,将小鼠分为空白对照组(经腹腔注射0.9%生理盐水,0.2 ml/d)、氨氯地平组[用氨氯地平片剂灌胃,10 mg/(0.1 ml·10 g体重·d)]、5-Fu组[分别于接种后第3、6、9天,经腹腔注射5-Fu溶液,10 mg/(1 ml·10 g体重·d)]及联合用药组(氨氯地平及5-Fu的给药方式与剂量同氨氯地平组及5-Fu组),每组10只(雌雄各半),连续给药10 d,次日断颈处死小鼠,称瘤重,并计算肿瘤生长抑制率。结果氨氯地平组、5-Fu组及联合用药组的细胞生长抑制率均随药物浓度增加逐渐上升,且呈剂量-时间依赖性。联合用药组与氨氯地平组及5-Fu组比较,H22细胞的凋亡率明显升高(P<0.05);Bcl-2表达水平明显降低,Bax的表达水平明显升高(P<0.05);小鼠体内瘤重明显降低(P<0.001),肿瘤生长抑制率明显升高(P<0.01)。结论氨氯地平与5-Fu联合用药对体内H22细胞的抑制作用明显强于各单独用药组,具有协同作用,为氨氯地平作为一种化疗增敏剂用于肿瘤的临床治疗提供了实验依据。

Objective To investigate the inhibitory effect of amlodipine combined with 5-fluorouracil（5-Fu） on murine hepatoma H22 cells. Methods H22 cells were treated with amlodipine at final concentrations of 3. 125,6. 25,12. 25, 25 and 50 μmol / L and 5-Fu at final concentrations of 12. 5,25,50,100 and 200 μg / ml,alone or in combination,for 24,48 and 72 h separately. The inhibitory effect on growth of H22 cells was determined by MTT assay. The apoptosis rate of H22 cells 48 h after treatment was determined by flow cytometry,while the expression levels of Bcl-2 and Bax by immunocytochemical assay. Mice were inoculated s.c. with 1. 0 × 107cells / ml of H22 cells,0. 2 ml for each,and divid- ed into four groups on the 2nd day,10 for each（5 male and 5 female）. The mice in blank control group were injected i.p. with physiological saline,0. 2 ml / 10 g body weight once a day,while those in amlodipine group were treated with amlodipine tablet by gavage,10 mg /（0. 1 ml · 10 g bodyweight · d）,those in 5-Fu group were injected i.p. with 5-Fu solution on days 3,6 and 9 after inoculation with H22 cells,10 mg（1 ml · 10 g bodyweightod）,and those in combi- nation group were treated with amlodipine and 5-Fu by the above-mentioned method. The mice in various groups were treated for 10 d then killed,of which the tumors were weighed,and the inhibiting rate of tumor growth was calculated. Results The inhibitory rates on cell growth in amlodipine,5-Fu and combination groups increased gradually in dose- and time-dependent modes. The apoptosis rate of H22 cells in combination group increased significantly as compared with those in amlodipine and 5-Fu groups（P 0. 05）,while the Bcl-2 expression level decreased significantly,the Bax expression level increased significantly（P 0. 05）,tumor weight decreased significantly（P〈 0. 001）,and the inhibiting rate to tumor growth increased significantly（P〈 0. 01）. Conclusion The inhibitory effect of amlodipine combined with 5-Fu on H22 cells in vivo was significantly stronger than those of amlodipine and 5-Fu alone, indicating a synergic effect, which provided an experimental basis for application of amlodipine as a sensitizer for chemotherapy of tumors.