芹菜素对自杀基因治疗系统杀伤人黑色素瘤细胞株A375的增效作用

Synergistic Effect of Apigenin on Suicide Gene Therapy of Human Melanoma Cell Line A375

目的研究芹菜素对单纯疱疹病毒1型胸苷激酶/更昔洛韦(HSV1-tk/GCV)自杀基因杀伤人黑色素瘤细胞株A375的增效作用及其机制。方法采用四甲基偶氮唑盐(MTT)法检测芹菜素对A375(tk^-)细胞生长的影响,及自杀基因治疗系统联合芹菜素对含20%A375-tk/GFP(tk^+)细胞的tk^+和tk^-混合细胞的杀伤效应的影响,流式细胞仪荧光示踪法分析缝隙连接通讯(GJIC)功能、细胞凋亡变化。结果芹菜素作用A375细胞24,48,72 h的半数抑制量(IC_(50))分别为86.34,25.81,11.16μmol·L^(-1),选用2.5~20μmol·L^(-1)芹菜素进行后续试验;5,10,20μmol·L^(-1)芹菜素联合GCV组对肿瘤细胞的抑制率明显比芹菜素组和GCV组高(P<0.01),协同性分析金正均Q值分别为2.03,1.43和1.36;2.5,5,10μmol·L^(-1)芹菜素处理A375细胞48 h后,各组绿色荧光细胞比值为:0.333±0.003,0.393±0.006,1.105±0.094,芹菜素各组比值均明显高于空白对照组(P<0.01);芹菜素联合GCV各组,与空白对照组、GCV组、芹菜素各组比较,可以增加细胞晚期凋亡率,趋势与MTT结果一致。结论芹菜素可以提高tk^+和tk^-混合细胞对GCV的敏感性,具有协同增强自杀基因系统杀伤效应的作用,其机制可能与芹菜素能够促进A375细胞GJIC功能及诱导细胞凋亡有关。

Objective To observe the enhancing effect and synergistic mechanism of apigenin on HSV1-tk/GCV suicide gene therapy for human melanoma cell line A375. Methods Methyl thiazolyl tetrazolium（MTT） assay was adopted to observe the inhibitive effect of apigenin on the proliferation of A375 cells（tk^-）,as well as the anti-tumor effects of suicide gene therapy combined with apigenin on tk~＋ tk^- mixed cells with 20 % A375-tk/GFP（tk~＋）. The gap junction intercellular communication（GJIC）function and apoptosis of A375 cells were measured by flow cytometry and fluorescent trace method. Results IC_（50） of apigenin on A375 cells after treatment for 24, 48, 72 h were 86.34,25.81, 11.16 μmol·L^（-1）respectively. The concentrations of 2.5~20 μmol·L^（-1） of apigenin were selected for the subsequent tests. The anti-tumor effect of 5, 10, 20 μmol·L^（-1） apigenin combined with GCV groups had higher tumor-inhibition rates than apigenin groups and GCV group（P 0.01）,and the Jin＇s Q values of synergistic analysis were 2.03, 1.43, 1.36. After A375 cells were treated with 2.5, 5, 10 μmol·L^（-1） apigenin for 48 h, the green fluorescent positive cell ratio of each group was 0.333 ±0.003, 0.393 ±0.006, 1.105 ±0.094, respectively, and apigenin groups had higher ratios than the blank control group（P 0.01）. Compared with the blank control group,GCV group and apigenin groups,the apoptotic rate of A375 cells in apigenin combined with GCV groups at late period was increased,and the trend was consistent with that showed by MTT results. Conclusion Apigenin can improve the sensitivity of tk~＋ tk^- mixed cells to GCV,and has synergistic action on enhancing the anti-tumor effect of suicide gene therapy system. Its mechanism may be related to the promotion of GJIC function and apoptosis of A375 cells.