小鼠腹水型肝癌淋巴道高转移细胞系HCa-P/L6细胞模型的建立及其药物敏感性的评价

Establishment and drug sensitivity evaluation of murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(HCa-P/L6)

关键词:  HCa-P  HCa-P/L6细胞模型  姜黄素  细胞周期

Key words:  HCa-P  HCa-P/L6 cell line  Curcumin  cell cycle

摘要:

［目的］建立小鼠腹水型肝癌淋巴道高转移细胞系HCa-P/L₆细胞模型并应用姜黄素对该模型的药物敏感性进行评价。［方法］将小鼠腹水型肝癌淋巴道低转移细胞株(HCa-P)经615小鼠腹中线皮下接种的方法获得淋巴结转移瘤1代细胞HCa-P/L1，再经小鼠淋巴系统筛选5次，筛选高转移细胞系HCa-P/L₆。用15～240 μmol·L^-1姜黄素(Curcumin, Cur)分别处理人肝癌细胞系(SMC7721和HepG2)和小鼠腹水型肝癌细胞(HCa-P/L6和HCa-P) 48 h，以MTT法考察4种细胞的药物敏感性。以非细胞毒性最大剂量Cur作用于HCa-P/L₆和HCa-P，以生长曲线和群体倍增时间评估药物对细胞生长的影响，以流式细胞仪检测药物对细胞周期分布的影响，考察两株小鼠腹水型肝癌细胞与姜黄素作用敏感性的差异。［结果］从HCa-P中筛选出具有多部位转移特性的淋巴道高转移细胞系HCa-P/L₆，转移率为100%。Cur明显抑制小鼠腹水型肝癌和人肝癌细胞的增殖，并呈剂量依赖性（方差分析P<0.05）。在30～120 μmol·L^-1，Cur对小鼠肝癌的抑制作用强于对人肝癌的抑制作用；在60～240 μmol·L^-1范围内，Cur对HCa-P/L₆的抑制作用强于对HCa-P。在非细胞毒性最大剂量15 μmol·L^-1Cur作用下，HCa-P/L₆和HCa-P的生长受到抑制（t检验P<0.05），并且呈时间依赖性（方差分析P<0.01），HCa-P/L₆和HCa-P的群体倍增时间延长（卡方检验P<0.01），Cur对HCa-P/L₆的抑制作用大于对HCa-P；细胞周期被重新分布，HCa-P/L₆被阻滞在S期，HCa-P被阻滞于S期和G2/M期。［结论］从HCa-P中筛选出了淋巴道高转移细胞系HCa-P/L₆。小鼠腹水型肝癌比人肝癌对姜黄素作用敏感，而且高转移细胞株系HCa-P/L₆较低转移细胞株HCa-P对Cur更为敏感。HCa-P/L₆为肿瘤淋巴道转移机制和中药活性成分筛选的研究提供了一个新的敏感模型。

Abstract:

［Objective］A new murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(HCa-P/L₆) was established and its sensitivity to curcumin was evaluated.［Methods］Murine ascites hepatocarcinoma cell strain with low lymphatic metastatic potential(HCa-P) was subcutaneously inoculated into the medioventral line of a mouse 615 and the first passage metastatic tumor cells of inguinal lymph node(HCa-P/L1) were obtained, and then HCa-P/L1was screened by the route of mouse lymphatics for five times. The sensitivity of two murine ascites hepatocarcinoma cell lines(HCa-P and HCa-P/L₆) and two human hepatocarcinoma cell lines(SMC7721 and HepG2) to curcumin were studied by the use of MTT assay after theses cells had been pretreated by curcumin at 15～240 μmol/L for 48 h. Treated by the maximum uncytotoxicity dose of curcumin, the effects of curcumin on cell proliferation and cell cycle of HCa-P/L₆and HCa-P were observed by making growth curve and the colony doubling times as well as flow cytometery(FCM).［Results］IA novel murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(HCa-P/L₆) spreading to lymphatic nodes of multiple sites in mice was screened from HCa-P and lymphatic node spreading rate is 100%. Curcumin exhibit evidently growth inhibition on both murine ascites and human hepatocarcinoma cell lines in dose-dependent manner(χ² test P<0.05). At concentration of 30～120 μmol·L^-1,curcumin had more inhibiton on murine ascites hepatocarcinoma cell lines than on human hepatocarcinoma cell lines. At concentration of 60～240 μmol·L^-1, curcumin had more inhibiton on HCa-P/L₆than on HCa-P with IC5051.48 μmol·L^-1in HCa-P/L6and 90.87 μmol·L^-1in HCa-P. Treated by the maximum uncytotoxicity dose of curcumin 15 μmol·L^-1for 7 days, the growth of HCa-P/L₆and HCa-P was inhibited( t test P<0.05) in time-dependent manner( χ² test P<0.01) and the colony doubling times of HCa-P/L₆and HCa-P were prolonged(P<0.01), and curcumin had more inhibiton on HCa-P/L₆than on HCa-P( t test P<0.05). Treated by curcumin 15 μmol·L^-1for 48 h, the cell cycle was redistributed with HCa-P/L₆being arrested in S phase while HCa-P in S and G2/M phases.［Conclusions］A novel murine ascites hepatocarcinoma cell line with high lymphatic metastatic potential(HCa-P/L₆) was screened from HCa-P. Two murine ascites hepatocarcinoma cell lines are more sensitive to curcumin than two human hepatocarcinoma cell lines and HCa-P/L₆is more sensitive than HCa-P. HCa-P/L₆is a novel sensitive cell line model for investigating the mechanisms of the lymphatic spread of a tumor and the effect of medicine on cells.