摘要
目的 将多药耐药基因 (mdr1)转入细胞因子诱导的杀伤 (cytokine inducedkiller,CIK)细胞 ,观察其是否产生耐药性并且保持肿瘤杀伤活性。方法 用IFN γ ,CD3单抗 ,IL 2 ,IL 1等细胞因子体外诱导外周血单个核细胞获得CIK细胞。采用电穿孔方法将mdr1基因表达质粒pHamdr转入CIK细胞。转染后 72h提取细胞总RNA ,DNA酶消化残留质粒DNA后进行RT PCR ,鉴定mdr1基因表达 ;流式细胞仪检测细胞膜耐药蛋白 (P gp)表达。四唑蓝比色法 (MTT)检测转基因后CIK细胞对阿霉素和秋水仙碱的耐药性 ;同时检测转染前后CIK细胞对MCF7细胞 (人类乳腺癌细胞系 )的杀伤活性变化。结果 转染mdr1后的CIK细胞mdr1mRNA阳性 ,P gp阳性的CIK细胞为 2 1%～ 37% (平均 2 7% )。转染后CIK细胞获得了多药耐药性 ,对阿霉素的IC50 值较转染前升高了 2 2 .3～ 4 5 .8倍 ,对秋水仙碱的IC50 值升高了 6 .7～ 11.35倍。转染前后CIK细胞对MCF7肿瘤细胞的杀伤活性无明显变化 (P >0 0 5 )。结论 CIK细胞转入mdr1基因后获得了多药耐药性 ,转染后的CIK细胞仍然保持原有的肿瘤细胞杀伤活性。
Objective To investigate whether the cytokine-induced killer (CIK) cells could acquire multidrug resistance and maintain the original cytotoxic activity after multidrug resistance (mdr1) genes transfection. Methods CIK cells were generated from peripheral blood cultured with IFN-γ, CD 3 monoclonal antibody, IL-2, IL-1 and transfected with a plasmid (pHamdr) containing human mdr1 gene via electroporation. RT-PCR method was used to assay mRNA expression of mdr1 gene in transfected CIK cells, flow cytometry with anti-P-gp monoclonal antibody to detect P-glycoprotein (P-gp) expression on CIK cells membrane,and MTT assay to compare both the multidrug resistance to doxorubicin and colchicines and cytotoxic activity to human mammary cancer cell line MCF7 between transfected and non-transfected CIK cells. Results mdr1 expression was detected in the transfected CIK cells. There was a strong expression of P-gp on the transfected CIK cells and the percentages of P-gp positive cells were 21%～37%(average 27%). The IC 50 of transfected CIK cells to doxorubicin was 22.3～45.8 times and 6 7～11.35 times to colchicines of those of non-transfected CIK cells. The cytotoxic activity to MCF7 remained unchanged(P>0.05). Conclusion It demonstrated that CIK cells transfected with mdr1 gene via electroporation could express multidrug resistance successfully without changes of cytotoxic activity.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2003年第12期617-620,共4页
Chinese Journal of Hematology
基金
国家自然科学基金资助 ( 3 9970 13 1)
北京市自然科学基金资助 ( 70 3 2 0 2 8)
