摘要
目的探讨携带多药耐药基因1(multidrug resistance gene 1,mdr1)反义RNA的重组腺病毒载体靶向逆转甲胎蛋白阳性(AFP+)的肝癌多药耐药细胞HepG2R的疗效及作用机制。方法分别构建携带AFP启动子和mdr1基因反义核苷酸片段的重组腺病毒载体Adeno-asmdr及携带AFP启动子和增强绿色荧光蛋白基因的重组腺病毒载体Adeno-EGFP,将Adeno—EGFP转染人正常肝细胞L02(AFP-)、人宫颈癌细胞HeLa(AFP-)及HepG2(AFP+)细胞,检测增强绿色荧光蛋白基因在各细胞的转录水平;将Adeno-asmdr转染HepG2R细胞,Western blot检测不同时间P—gp170的表达,末端脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末端标记法检测HepG2R细胞凋亡,流式细胞术检测HepG2R细胞在不同药物作用下细胞周期、凋亡率。结果增强绿色荧光蛋白基因在AFP阳性的HepG2细胞可得到显著转录,而在L02细胞和HeLa细胞,其转录减少,显示了该载体的良好转录活性以及靶向特异性。Adeno—asmdr转染HepG2R细胞后,HepG2R细胞P—gp170表达明显减弱,HepG2R细胞凋亡增加,HepG2R细胞对多种化疗药物的耐受能力明显下降,细胞出现显著的周期阻滞,大量细胞被阻滞于S期和G0/M期,凋亡细胞比例增加。结论实验构建的Adneo—asmdr重组腺病毒载体可在AFP阳性HepG2R细胞内特异靶向性表达目的基因,并可有效降低mdr1基因产物P—gp170的表达,从而达到对HepG2R细胞多药耐药的逆转作用。
Objective To investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells. Methods An adenovirss vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry. Results EGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adcno-asmdr, and the expression of P-gpl70 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chrmicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase. Conclusion The recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2009年第8期594-598,共5页
Chinese Journal of Hepatology
基金
重庆市卫生局重点课题(渝卫01-1-018)
关键词
癌
肝细胞
腺病毒
反义技术
多药耐药基因1
Carcinoma, hepatocellular
Adenovirus
Antisense technology
Multidrug resistance gene 1