摘要
目的 构建携带多药耐药mdr1全长基因的真核表达载体并研究其在人肝癌细胞HepG2 中的表达。方法 双酶切质粒pHaMDR1,获取含mdr1全长cDNA片断,将此片段定向克隆到真核表达载体PCI neo多克隆位点,经脂质体法转染HepG2细胞,G418筛选稳定的细胞系HepG2/mdr1,PCR检测mdr1 特异片段,RT PCR 检测HepG2/mdr1细胞mdr1 mRNA表达,流式细胞仪检测HepG2/mdr1 细胞P gp的含量。结果 成功构建携带mdr1全长基因的真核表达载体,并在HepG2 细胞中表达,形成稳定的细胞系HepG2/mdr1,其mdr1 mRNA及P gp的含量较未转染该载体的HepG2细胞显著增加。结论 用真核表达载体将mdr1 全长基因导入人肝癌细胞HepG2能够建立高效、稳定的多药耐药细胞系,为进一步研究多药耐药机理提供理想的细胞模型。
Objective To construct an mdr1 expression vector and detect its expression in HepG2 cells in vitro . Methods The 4.5-kb mdr1 cDNA was obtained from the plasmid pHaMDR1 cloned into the PCI-neo mammalian expression vector, which was later transferred into human hepatocarcinoma cell line HepG2 by liposome. Then the HepG2 cells resisting G418 were clustered and proliferated,and the specific fragment of mdr1 cDNA, mRNA and the P-gp in these HepG2 cells were detected by means of PCR, RT-PCR and FCM respectively. Results The mdr1 expression vector was constructed successfully,and the stable multidrug resistance(MDR) hepatocarcinoma cell line (HepG2/mdr1) was developed as well. The outcome of PCR analysis showed that the specific fragment of mdr1 cDNA could be found in HepG2/mdr1 cells, but not in the nontransfection HepG2 cells. Furthermore,the content of the specific fragment of mdr1 mRNA and the expression of P-gp in HepG2/mdr1 cells were (59.7±7.9)% and (12.5±5.45)% respectively, the corresponding value in HepG2 cells were (16.9±3.2)% and (4.63±2.59)% respectively. The difference was statistically significant ( P <0.05). Conclusion It is praticable to develop MDR hepatocarcinoma cell line by transferring mdr1 cDNA into HepG2 cells, which is useful in the research of MDR mechanism.
出处
《中国普外基础与临床杂志》
CAS
2005年第3期254-257,共4页
Chinese Journal of Bases and Clinics In General Surgery
基金
国家自然科学基金资助(编号: 30170925)~~
