摘要
目的:探讨人铜离子转运蛋白1(humanc opper transporter1,hCTR1)与卵巢癌细胞对铂类药物耐药的关系。方法:构建重组反转录病毒载体pBABEpuro-hCTR1,转染包装细胞后感染人卵巢癌细胞株SKOV-3,用嘌呤霉素筛选得到稳定表达hCTR1的pBABEpuro-hCTR1/SKOV-3细胞。应用实时荧光定量.PCR(real time fluorogentic quantitative PCR,RFQ-PCR)和Western印迹法检测pBABEpuro-hCTR1/SKOV-3细胞中hCTR1mRNA和蛋白的表达,MTT法检测顺铂(cisplatin,DDP)对pBABEpuro/SKOV-3和pBABEpuro-hCTR1/SKOV-3细胞的增殖抑制率,FCM检测DDP对上述细胞周期的影响,电感耦合等离子质谱法(ion-coupled plasma-mass spectroscopy,ICP-MS)测定细胞内的铂含量。结果:成功构建了hCTR1高表达的SKOV-3稳定细胞株。DDP对pBABEpuro-hCTR1/SKOV-3细胞的IC50值为(18.97±1.24)μmol/L,对pBABEpuro/SKOV-3细胞的IC50值为(29.35±2.12)μmol/L(P<0.01)。DDP对pBABEpuro-hCTR1/SKOV-3细胞的S期阻滞作用大于pBABEpuro/SKOV-3细胞。pBABEpuro-hCTR1/SKOV-3细胞对DDP的摄入能力大于pBABEpuro/SKOV-3细胞。结论:hCTR1参与卵巢癌细胞对DDP的转运,其高表达可增强SKOV-3细胞对DDP的摄入能力和敏感性。
Objective：To investigate the correlation between human copper transporter 1 （hCTR1） and cisplatin（DDP）resis-tance in ovarian carcinoma.Methods：The pBABEpuro retrovirus vector containing cDNA encoding hCTR1 was constructed by recombinant DNA technology. The packaging cells 293T were transfected with this recombinant plasmid by liposome-based transfection method. The virus supernatant was collected and then infected SKOV-3 cells. The stable integrant was selected by puromycine screening. The expressions of hCTR1 mRNA and protein were identified by real time fluorogentic quantitative PCR and Western blotting,respectively. The effect of DDP on proliferation of pBABEpuro-hCTR1/SKOV-3 cells was determined by MTT assay. The effect of DDP on cell cycle and intracellular platinum was investigated by flow cytometry and ion-coupled plasma-mass spectroscopy （ICP-MS）,respectively. Results：The SKOV-3 clone stably and effectively expressing hCTR1 was successfully selected. Compared with control pBABEpuro/SKOV-3 cells,PBABEpuro-hCTR1/SKOV-3 cells were more sensitive to DDP[IC50：（18.97±1.24） μmol/L vs （29.35±2.12） μmol/L,P〈0.01]. The blocking effect of DDP on S phase was stronger in pBABEpuro-hCTR/SKOV-3 cells than that in control cells. pBABEpuro-hCTR1/SKOV-3 cells accumulated more platinum than pBABEpuro/SKOV-3 cells.Conclusion：hCTR1 is involved in the transportation of DDP by ovarian cancer cells. Overexpression of hCTR1 increases the intracellular accumulation of DDP and enhances the sensitivity of SKOV-3 cells to DDP.
出处
《肿瘤》
CAS
CSCD
北大核心
2009年第10期934-939,共6页
Tumor
基金
上海市科委基础研究重点项目(编号:05JC14012)
关键词
卵巢肿瘤
抗药性
肿瘤
顺铂
人铜离子转运蛋白
Ovarian neoplasms Drug resistance neoplasm Cisplatin Human copper transporters
