导入MDR1建立肝癌耐药细胞系及其动物实验研究

Establish hepatocellular carcinoma multidrug resistance cell line by transferring the MDR1 cDNA

目的探讨利用转染多药耐药基因(MDR1)方法建立表肝癌耐药细胞系,为深入研究肝癌多药耐药现象提供理想的细胞模型。方法将前期构建表达mdr1cDNA转染质粒PCI/mdr1转染人肝癌细胞株HepG2细胞,采用G418和阿霉素短暂诱导,筛选出稳定的耐药细胞株HepG2/mdr1;利用RT-PCR、FCM及体内外的耐药性试验对其进行功能检测。结果与其亲代细胞株HepG2比较,HepG2/mdr1细胞中mdr1mRNA及P-gp表达明显增加,分别为(58.80%±11.80%vs 24.00%±5.80%)和(10.28±2.09 vs 3.70±1.06),对阿霉素和丝裂霉素的耐药性分别增加了35倍和125倍,体内耐药性也明显增加。结论利用转基因方法能够成功建立肝癌多药耐药细胞系。

Objective To establish a stable HCC MDR cell line on the basis of transgenic mdr1.Methods The 4.5-kb mdr1 cDNA was transferred into human hepatocarcinoma cell line HepG2 by the PCI-neo mammalian expression vector and the transfected HepG2 cells resisting G418 were proliferated.Then the mdr1 mRNA and P-gp in these HepG2 cells were detected by the means of RT-PCR and FCM respectively.The accumulation of the daunorubicin was determined by FCM simultaneously.The nude mice model of grafting tumor was established by injecting subcutaneously HepG2/mdr1 cells in the right axilla and ADM was injected into peritoneal cave when the tumor diameter reached 5mm.The size and growth inhibition of the tumor were evaluated.Results The MDR hepatocarcinoma cell line HepG2/mdr1 was developed and the mdr1 mRNA and P-gp in HepG2/mdr1 cells were（58.80±11.80）%and（10.28±2.09）respectively,compared with（24.00±5.80）% and（3.70±1.06）in HepG2 cells.The resistance to ADM and MMC increased 35 and 125 times respectively.In the nude mice HCC model,after ADM therapy,the mean size of HepG2 cell tumors was obviously smaller than HepG2/mdr1 cell tumors,the difference was significant.Conclusions The approach using the transfer of mdr1 cDNA may be applicable to the development of MDR hepatocarcinoma cell line which would provide the experimental basis of MDR research.