人肝癌BEL-7402/5-FU多药耐药细胞株的建立及其生物学特性观察

目的:建立人肝癌BEL-7402/5-FU多药耐药细胞株。方法:采用体外低浓度梯度递增联合大剂量间断冲击的诱导方法建立5-FU获得性BEL-7402/5-FU多药耐药细胞株。MTT法检测耐药细胞株对多种化疗药物的交叉耐药性。观察其细胞形态学、生长曲线、倍增时间、平板克隆形成率、贴壁率、细胞周期分布、染色体核型以及裸鼠致瘤性。流式细胞术检测阿霉素在亲本及耐药细胞株内的积聚量。免疫细胞化学法检测胸苷酸合酶在耐药细胞内的表达。结果:成功建立人肝癌BEL-7402/5-FU多药耐药细胞株模型。该耐药细胞对阿霉素、长春新碱、奥沙利铂及甲氨蝶呤均有不同程度的交叉耐药性,但对羟基喜树碱仍较敏感。体外培养见细胞趋向群集性生长。耐药细胞株倍增时间较亲本细胞株长,克隆形成率则较亲本细胞株低,差异有统计学意义。耐药细胞贴壁率在23、h明显低于亲本细胞株,其G0/G1期细胞分布比率较低而S期比率明显增加。阿霉素在耐药细胞株内的积聚量低于亲本细胞株,耐药细胞株胸苷酸合酶的蛋白表达量较亲本细胞株明显增强。结论:人肝癌BEL-7402/5-FU多药耐药细胞株可以成为研究5-FU获得性耐药机制及开展多药耐药逆转剂筛选较好的体外模型。

Establishment of a multidrug-resistant cell line BEL-7402/5-FU of human hepatocellular carcinoma and its biological characteristics

Objective: To establish a multidrug-resistant cell line BEL7402/5-FU of hepatocellular carcinoma(HCC).Methods: BEL-7402/5-FU was induced by pulse therapy combined with continuous stepwise exposure to 5-fluorouracil in vitro.MTT assay was used to determine its multidrug resistance(MDR).Biological characteristics of the BEL-7402/5-FU cell line were observed including morphological changes,cell growth curve,population doubling time,plate cloning efficiency,adherence rate,cell cycle distribution,chromosome and tumorigenicity.Accumulation amount of adriamycin(ADM) in cytoplasm was measured by flow cytometry.The protein expression of thymidylate synthase(TS) was evaluated by immuno-cytochemical method.Results: The acquired MDR cell line of BEL-7402/5-FU was established successfully.The BEL-7402/5-FU cells showed cross-resistance to ADM,vincristine(VCR),methotrexate(MTX) and oxaliplatin(OHP),whereas still sensitive to hydroxycamptothecin(HCPT).The BEL7402/5-FU cells tended to grow in clusters in vitro.It was found that the population doubling time of BEL-7402/5-FU cells was longer than that of its(parental) cells.The plate cloning efficiency and the adherence rate of BEL-7402/5-FU cells at the 2nd and 3rd hour were both lower than those of the parental cells.The distributing proportion of BEL-7402/5-FU cells in G_(0)/G_(1) phase was less than that of the parental cells,whereas the distributing proportion of BEL-7402/5-FU cells in S phase was higher than that of the parental cells.The accumulation amount of ADM in cytoplasm of BEL-7402/5-FU cells was significantly lower while the expression level of TS protein of which was highly(up-regulated) as compared with those of the parental cells.Conclusion: Establishment of the human HCC cell line BEL-7402/5-FU might be beneficial to the studies of 5-Fluorouracil acquired MDR mechanisms and the selection of reversal modifiers.