两种方法建立的人肝癌细胞多药耐药模型的比较

Comparison between two human multi-drug resistant hepatocarcinoma cell lines established via two different methods separately

目的 比较药物大剂量冲击法和浓度梯度递增法建立的人肝癌多药耐药模型的生物学特性。方法 分别采用药物大剂量冲击法和浓度梯度递增法建立人肝癌阿霉素(adriamycin,ADM)多药耐药细胞亚系HepG2 /ADMs和HepG2 /ADMi。MTT法检测两种细胞对多种化疗药的敏感性,细胞计数法绘制生长曲线并用公式法计算倍增时间,流式细胞术检测细胞周期以及细胞对药物的摄入和外排。并将细胞接种于裸鼠,观察成瘤率及瘤体大小。结果 以含1μg/mLADM的培养液进行筛选,3个月共筛选4次,所得细胞亚系命名为HepG2 /ADMs,其对ADM的耐药指数为30.5。浓度梯度递增法诱导3个月后,细胞可以在含0.4μg/mLADM的培养液中正常生长增殖,对ADM的耐药指数为51,将该细胞亚系命名为HepG2 /ADMi; MTT法检测显示,两种细胞系不仅对ADM耐药,而且对其它化疗药也有抗性,呈现多药耐药特性,但HepG2 /ADMs对药物的敏感性普遍高于HepG2 /ADMi。与HepG2 /ADMi相比,HepG2 /ADMs的倍增时间相对较短,G0 /G1期细胞相对较少,G2 /M期细胞相对较多,细胞对ADM的摄入较多,外排较少,表现为药物的滞留率较高。动物实验发现HepG2 /ADMs的成瘤率明显高于HepG2 /ADMi(90% vs3.33% ),且肿瘤生长速度较快,体积较大。

Objective To compare biological characteristics of two human multi-drug resistant hepatocarcinoma cell lines selected/induced by pulse drug exposure and continuous stepwise exposure, respectively. Methods Human hepatocarcinoma cell line HepG 2 were exposed to high concentration of adriamycin transiently and stepwise increasing concentrations of adriamycin continuously, respectively, where the corresponding new sublines were designated HepG 2/ADMs and HepG 2/ADMi, respectively. The MTT assay was used to evaluate drug sensitivity. Cell counting was performed by drawing cell growth curves. Doubling time for cell line was calculated according to Patterson formula. Flow cytometric assay was employed to examine cell cycle and to determine drug influx and efflux. At last the cells were subcutaneously injected into nude mice. Tumorigenicity of cells was observed and tumor volume was measured. Results Totally 4 high-concentration-drug-exposures(1μg/ml ADM) were carried out within 3 months and the selected subline HepG 2/ADMs presented a resistance index of 30.5 against ADM. The other subline HepG 2/ADMi resulting from 3 months′ stepwise drug exposure exhibited more resistant feature. It could normally grow and propagate in medium supplemented with 0.4μg/ml ADM. The MTT assay showed that the two sublines were resistant to not only ADM, but also other chemotherapy agents manifesting multidrug resistant characteristic. HepG 2/ADMs was more sensitive to most drugs than HepG 2/ADMi. And doubling time for HepG 2/ADMs was shorter. The number of cells for HepG2/ADM in the G 0/G 1 phase of the cell cycle was lower, whereas the number of cells in the G 2/M was higher. ADM influx was augmented while ADM efflux decreased, so that the retention rate was higher. The transplantation of the sublines in nude mice demonstrated that HepG 2/ADMs had a much higher tumor-forming rate than did HepG 2/ADMi(90% vs 3.33%). Tumors derived from HepG 2/ADMs grew rapidly. Conclusion Multidrug resistant subline selected by pulse exposure is very different from that induced by stepwise exposure. The former is closer to clinical chemoresistance of tumor.