白介素-4基因修饰对儿童肝母细胞瘤多药耐药的逆转及其机制研究  

Reversion of multidrug resistance by interleukin-4 gene transfer in hepatoblastoma cell line cells

作　　者：付劲蓉[1] 李成荣[1] 杨锡强[1] 周雅德[1] 

机构地区：[1]重庆医科大学儿科研究所

出　　处：《中华儿科杂志》2001年第11期650-653,共4页Chinese Journal of Pediatrics

基　　金：国家自然科学基金 (3 9470 73 3 );深圳市卫生厅科研基金资助

摘　　要：目的　探讨白介素 4 (IL 4 )基因修饰对儿童实体瘤多药耐药 (MDR)的逆转作用及可能机制。方法　用逆转录病毒载体将人IL 4基因导入人肝母细胞瘤细胞系HepG2 ,采用MTT生物活性法检测瘤细胞对化疗药物的敏感性。间接免疫荧光分光光度分析瘤细胞P 糖蛋白 (P gp)的表达率。微量荧光分光光度法分析瘤细胞内阿霉素 (ADM)聚集量。结果　IL 4基因修饰显著降低瘤细胞对种化疗药物的半数抑制浓度 (IC5 0 ) ,表现出MDR逆转效应 (耐药逆转倍数在 6～ 32倍数间 )及明显增加化疗药物在瘤细胞内聚集 ,同时IL 4基因修饰瘤细胞P gp阳性表达率 (5± 0 6 % )较野生型及空载体修饰细胞 (98 5± 0 5 %、97 0± 1 0 % )明显减低。IL 4基因修饰对瘤细胞的MDR逆转作用可为PKC激活剂TPA所阻断。IL 4基因修饰瘤苗及其培养上清与野生型细胞共培养 ,也可使野生型瘤细胞产生MDR逆转效应。结论　IL 4基因修饰瘤苗可通过自分泌、旁分泌形式逆转瘤细胞MDR ,此作用可能与其抑制PKC活性及下调PObjective To investigate the reversing effect of interleukin 4 (IL 4) gene transfer and its mechanism in multidrug resistance (MDR) of children′s solid tumor cells and to find the reasons for combined use of the tumor vaccine modified by IL 4 gene transfer and chemotherapeutic agents. Methods Retroviral vector (PL IL 4 SN) was selected to introduce human IL 4 gene into children′s hepatoblastoma cell line (HepG 2) cells. Reverse transcriptase polymerase chain reaction (RT PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the expression of IL 4 in IL 4 gene transferred cells and sorted out the tumor cell clones which could stably secret about 600 800 pg/(106 cells.24 h) of IL 4. MTT assay was used to detect the sensitivity of these tumor cells to all sorts of chemotherapeutic agents such as vincristine (VCR), adriamycin (ADM), mitomycin (MMC) and etoposide (VP 16). Indirect immuno fluorescence staining by flow cytometry was used to evaluate the expression of p glycoprotein (p gp), which is one of the main multi drug resistant proteins in tumor cells. Microquantitative analysis with fluorometer was employed to detect the intracellular accumulation of ADM. Results Over expression of p gp (the positive rate of p gp expression were separately 98.5±0.5%, 97.0±1.0%) was found in the wild HepG 2 cells and the tumor cells could only be transferred with the vector not containing the IL 4 gene; and all the cells had a strong resistance to all the above agents. Otherwise the tumor cells transferred with IL 4 gene became greatly more sensitive to all the above agents with a very low positive rate of p gp expression (5 0±0.5%), the differences were significant (v=6, t =87.6, P <0.01) and the quantity of intracellular accumulation of ADM was greatly increased (v=10, t =3.2 10.6, P <0.05); the reversing effects on multi drug resistance could be inhibited by 100 ng/ml 12 0 tetradecanoyl phorbol 13 acetate (TPA), which is an activactor of protein kinase C

关 键 词：白细胞介素4 基因治疗法 儿童 肝母细胞瘤 多药耐药性 MDR 

分 类 号：R735.7[医药卫生—肿瘤] R730.5[医药卫生—临床医学]