一种肝癌多药耐药细胞系的建立及鉴定

One Way Inducing and Evaluating Multidrug Resistance Cell Line of Hepatocelluar Carcinoma Cell

目的建立肝癌细胞系HepG2的多药耐药细胞系HepG2/ADM,为研究肝癌细胞多药耐药创造前提条件。方法用0.01～2μg/mL的阿霉素(ADM)分级诱导HepG2细胞的多药耐药性,使其在含高浓度的ADM(2μg/mL)的DMEM培养基中保持90%的存活率,并能正常地传代、冻存和复苏;用MTT法分析ADM、5-氟尿嘧啶(5-FU)、丝裂霉素(MMC)和氨甲蝶呤(MTX)四种药物在其1Cmax,10Cmax,20Cmax和30Cmax时分别作用于HepG2和HepG2/ADM时的细胞生长抑制率;用免疫组化法观察细胞膜P-糖蛋白(P-gp)170的表达;用流式细胞仪(FCM)分析细胞膜P-gp170对进入细胞内药物罗丹明-123的泵出效果。结果HepG2/ADM细胞系表现了较强抗药活性;免疫组化法观察到HepG2/ADM细胞膜有较强P-gp170表达;FCM检测到HepG2/ADM细胞能有效泵出罗丹明-123,而HepG2细胞则不能有效泵出罗丹明-123。结论用ADM梯度诱导耐受法成功诱导出了HepG2细胞的多药耐药细胞系HepG2/ADM。

Objective rFo induee the hepatocelluar carcinoma cell （HCC） line HepG2 into multidrug resistance cell line HepG2/ADM. Methods The agent of ADM whose dose from 0. 01μg/mL to 2μg/mL was used the HCC line HepG2 into multidrug resistance cell line HcpG2/ADM,which have the 90% survival in the DMEM cuhure medium containing 2 Ltg/mL ADM in end-eoncentration and can reproduction,regain consciosness anti stored in liquid nitrogen. To abserve the HepG2 and HepG2/ADM cells antidrg activity to ADM, 5-flurouraeil （5- FU）,mitomycin （MMC） and methotrexate （MTX） at the dose of I C 10C 20Cmax 30Cmax the MIT＇s analyse was used. The immnnohistochemieal method was also used to appraise the P-glycoprotein 170 （P-gp 170） expression on the HepG2/ADM and HepG2 cell membrane.Flow eytometer was to investigate the concentration of Rhodamine-123 in the HepG2/ADM cell. Results The HepG2/ADM cell line have high efficient antidrug activity to the agents of ADM,5-FU,MMC and MTX, The P-gp 170 was deccted on the HepG2/ADM cell membrane with immunohistochemical method but not on HepG2 cell. Flow eytometer （FCM） showed the concen- tration of Rhodamine-123 in HepG2/ADM was very low anti there was obvious remainder of Rhodamine-123 in HepG2. Conclusion The HCC cell line HepG2 can be suecessfully induced into the multidrug resistanee eell HepG2/ADM with step by step inducion.