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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/DOXV和Bel-7402/DOXL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/DOXV和Bel-7402/DOXL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 Bel-7402 / Doxorubicin (DOX) model of human multidrug-resistant human hepatocellular carcinoma cell line (HCC) by increasing concentration gradient in vitro and inducing liver transplantation in nude mice. Methods The human hepatocellular carcinoma cell lines, Bel-7402 / DOXV and Bel-7402 / DOXL, were induced by increasing concentration gradient in vitro and induced by liver transplantation in nude mice respectively. The cells were observed by phase-contrast microscope. MTT assay The cell growth curve was calculated by cell count method and the doubling time was calculated by the formula method. The uptake and efflux of DOX and P-gp, multidrug resistance-related protein (MRP), glutathione S-transferase system (GSH / GST) expression. Results Both drug-resistant cell lines Bel-7402 / DOXV and Bel-7402 / DOXL produced cross-resistance to DOX and CDDP, with significant difference from the parental Bel-7402 IC50 (P <0.01) The doubling time was significantly longer at 65 h and 46 h, respectively. The DOX efflux rate of the two groups was significantly higher (81.06% and 66.56%, respectively) than that of the parental cells. The expression of P-gp and MRP in both drug- P <0.01), while the expression of GSH / GST had no significant change. Conclusion The drug resistance cell lines established by the two methods have stable drug resistance. Liver transplantation is more likely to simulate human hepatocellular carcinoma, which is a more ideal model with similar pharmacokinetic profiles of human hepatocarcinoma biology and anti-cancer drugs.