目的比较体外浓度梯度递增诱导和裸鼠肝脏移植诱导两种方法建立人肝癌多药耐药细胞系Bel-7402/多柔比星(Doxorubicin,DOX)模型的生物学特性。方法分别采用体外浓度梯度递增诱导和裸鼠肝脏移植诱导建立人肝癌多柔比星多药耐药细胞亚系Bel-7402/DOX_V和Bel-7402/DOX_L后,利用相差显微镜观察细胞,MTT法检测两种细胞的耐药性,细胞计数法绘制生长曲线并用公式法计算倍增时间,流式细胞仪测定细胞DOX的摄入和外排以及P糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、谷胱甘肽硫转酶系统(GSH/GST)的表达。结果两组耐药细胞Bel-7402/DOX_V和Bel-7402/DOX_L对DOX、CDDP均产生了交叉耐药性,较亲本Bel-7402 IC₅₀值差异有统计学意义(P<0.01);较亲本细胞的倍增时间明显延长,分别为65 h和46 h;较亲本的DOX外排率明显升高,分别为81.06%、66.56%;两组耐药细胞P-gp、MRP表达较亲本细胞显著提高(P<0.01),而GSH/GST的表达无明显变化。结论两种方法建立的耐药细胞系模型均有稳定的耐药性。肝脏移植法更能高度模拟人肝癌, 它是具有近似人肝癌生物学和抗癌药物动力学特征的较理想模型。

Objective To compare the biological characteristics of two types of human hepatocellular carcinoma multi-drug resistant cell sub-lines Bel-7402/DOX models established by two methods. Methods We established human hepatocellular carcinoma doxorubicin multi-drug resistant cell sub-lines models Bel-7402/DOXV and Bel-7402/DOXL by in vitro concentration gradient increased induction and nude mice liver-implanted induction, respectively. Phase contrast microscopy was used to observe the cells and MTT (methyl thiazolyl tetrazolium) method was used to detect drug resistance of the two different sub-lines of cells. The ingestion and excretion of cellular doxorubicin (DOX) and the expression of P-glycoprotein (P-gp), multi-drug resistance-sociated protein (MRP) and glutathione S-transfer enzyme system (GSH/GST) were detected by flow cytometry. Results The Bel-7402/DOXV and Bel-7402/DOXL generated cross-resistance to DOX and CDDP (cis-Diaminedichloroplatinum), they showed a significant difference in resistance to Bel-7402 IC50 value (P<0.01). The doubling times were significantly extended, compared with the parent cell line (39 h), and were 65 h (Bel-7402/DOXV) and 46 h (Bel-7402/DOXL). The excretion rates of DOX were significantly increased, compared with the parent cell (34.14%) line and were 81.06% (Bel-7402/DOXV) and 66.56% (Bel-7402/DOXL). Expressions of P-gp and MRP in the two groups of resistant sub-lines cells were significantly enhanced (P＜0.01). There was no significant variation in the expression of GSH/GST (P>0.05). Conclusions Stable resistance is involved in the resistant cell line model established by the two methods above. Liver implantation is a good simulation of human hepatocellular and proves to be an ideal model with characteristics similar to human hepatocellular biology and the pharmacokinetics of anticancer drugs.