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目的探讨携带多药耐药基因1(multidrug resistance gene1,mdr1)反义RNA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。方法将携带AFP启动子和EGFP基因的重组腺病毒载体Adeno-EGFP转染人正常肝细胞LO2(AFP-)、人宫颈癌细胞HeLa(AFP-)及HepG2(AFP+)细胞,检测EGFP基因在各细胞的转录水平,证实重组腺病毒载体的靶向性;将携带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转染HepG2R细胞和HepG2R裸鼠皮下移植瘤模型,检测Adeno-asmdr在体外和体内逆转HepG2R的活性。结果EGFP基因在AFP阳性的HepG2细胞可得到显著的转录,而在LO2细胞和HeLa细胞,其转录和表达量极少,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,mdr1转录水平下降;Rhodamine123在细胞内聚集量有所下降;在裸鼠皮下移植瘤实验中,经Adeno-asmdr+ADM处理组,移植瘤体积无增大,TUNEL法检测移植瘤内凋亡细胞增加,而经PBS和ADM处理组移植瘤体积明显增大(P<0.05),ADM处理组移植瘤中出现少量的凋亡细胞,而PBS处理组移植瘤未见凋亡细胞。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,有效降低mdr1基因产物P-gp170的表达,从而达到对HepG2R细胞MDR的逆转作用;结合化疗药物的作用,可以导致裸鼠皮下移植瘤细胞凋亡增加,阻止瘤体生长。
Objective To investigate the therapeutic effect and mechanism of multidrug resistance gene1 (mdr1) antisense RNA recombinant adenoviral vector targeting HepG2R on reversing AFP-positive hepatocarcinoma cell line HepG2R. Methods Human adenocarcinoma cell line AFP-, HeLa (AFP-) and HepG2 (AFP +) cells were transfected with recombinant adenovirus vector Adeno-EGFP carrying AFP promoter and EGFP gene. The Adeno-asmdr vector carrying AFP promoter and mdr1 antisense nucleotide fragment was transfected into HepG2R cells and HepG2R nude mice subcutaneously. Detection of Adeno-asmdr reverses HepG2R activity in vitro and in vivo. Results The EGFP gene was significantly transcribed in AFP positive HepG2 cells. However, the transcription and expression levels of EGFP gene in LO2 cells and HeLa cells were very low, indicating good transcriptional activity and targeting specificity. Adeno-asmdr transfection HepG2R cells, mdr1 transcriptional level decreased; Rhodamine123 in the cell aggregation decreased; in nude mice subcutaneously transplanted tumor experiments, the Adeno-asmdr + ADM treatment group, no increase in tumor volume, TUNEL method for the detection of transplanted tumors (P <0.05). The apoptotic cells were increased in the treated group and the apoptotic cells were increased in the PBS and ADM treated groups (P <0.05). A small amount of apoptotic cells were found in the transplanted tumors in the ADM treated group and no apoptotic cells in the PBS treated group. Conclusion The constructed Adneo-asmdr recombinant adenovirus vector can specifically target the AFP-positive HepG2R cells and effectively reduce the expression of mdr1 gene product P-gp170, so as to achieve the reversal of MDR in HepG2R cells. Combined with chemotherapy drugs The role of subcutaneous transplanted tumor cells can lead to increased apoptosis in nude mice to prevent tumor growth.