摘要
目的 建立人肝癌细胞系HepG2多药耐药模型并探讨低频脉冲式超声对其凋亡的影响及其机制。 方法 以人肝癌细胞株HepG2为研究对象,用阿霉素(ADM)浓度梯度递增诱导法,建立人肝癌细胞多药耐药模型(简称HepG2/ADM)。将研究对象分为HepG2/ADM、HepG2/ADM+ADM、HepG2/ADM+超声波、HepG2/ADM+ADM+超声波4组,以频率为0.8 MHz,声强为0.5 w/cm2,时间10 min的脉冲式超声波作用于研究对象。荧光显微镜观察细胞变化;DNA片段化分析检测染色体断裂情况;流式细胞仪检测细胞凋亡率。 结果 HepG2/ADM细胞对多种化疗药耐药,对阿霉素的耐药倍数为26,其耐药性与P-糖蛋白(P-GP)、多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)及谷胱甘肽转移酶(GST)的过表达相关。实验组经超声波作用10 min后,HepG2/ADM细胞死亡具有细胞凋亡的形态学特征;HepG2/ADM+ADM和HepG2/ADM+超声波组细胞凋亡率分别为3.47％和12.23％;HepG2/ADM+ADM+超声波联合治疗能显著增加细胞凋亡率,为18.81％,与对照组比较差异有显著性,t值分别为1.46、2.67、5.36,P<0.01。结论 人肝癌多药耐药细胞株HepG2/ADM具有多药耐药特性;低频脉冲式超声可诱导人肝癌多药耐药细胞株HepG2/ADM凋亡,联合化疗药物治疗,可显著增加细胞凋亡比例。
Objective To establish human hepatocellular carcinoma multidrug-resistance cell line (HepG2/ ADM) and to determine the effect of low-frequency pulse ultrasound (US) on MDR cells and investigate its mechanism. Methods Using gradual increase of adriamycin (ADM) concentrations in culture, an adriamycin-resistant human hepatocellular carcinoma cell sub line (HepG2/ADM) was established in vitro. HepG2/ADM cells were cultured in vitro and randomly divided into 4 groups: the control group (HepG2/ADM only), the group ADM by 1.0 μg/ml adriamycin for 1 h, the group US by low-frequency pulse ultrasound for 10 min, and the group US by low-frequency pulse ultrasound and treated with adriamycin simultaneously at same time. A sonication at a frequency of 0.8 MHz, was delivered with an intensity level of 0.5W/cm2, with continuous exposure of 10 min was applied. The ability of US to induce the apoptosis of MDR was evaluated by analyses of fluorescence microscopy, DNA fragmentation assay and flow cytometry assay. Results HepG2/Adm was resistant to many anti-tumor agents, and its IC50 of ADM was 26 times higher than that of parent cell line HepG2. Significant over expressions of P-gp, MRP, LRP and GSTs were detected. HepG2/ADM cells radiated by US had the typical characteristics of apoptosis. Compared with the control group (HepG2/ADM, 3.47%); the apoptosis rates were higher in US (12.23%). The therapeutic alliance of US with ADM for MDR cells, have a significant change in the ratio of apoptosis (18.81%, t=1.46-5.36, P < 0.01). Conclusion HepG2/ADM could have the biological characteristics of human multidrug-resistance cell line. The US sonication of 0.8 MHz could induce apoptosis of HepG2/ ADM cell in vitro, and could act synergistically with Adriamycin.
出处
《中华肝脏病杂志》
CAS
CSCD
2004年第2期95-98,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金(30200060)
