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纸质出版:2009
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梅英,杜伟,丁雄,陈晓光,简华刚,龚建平,刘长安.靶向重组腺病毒载体逆转肝癌细胞多药耐药的实验研究[J].陆军军医大学学报(原第三军医大学学报),2009,31(15):1462-1466.
MEI Ying,DU Wei,DING Xiong,CHEN Xiao-guang,JIAN Hua-gang,GONG Jian-ping,LIU Chang-an.Experimental study of the targeting recombinant adenoviral vector in reversing the mdr hepatocarcinoma cells[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(15):1462-1466.
梅英,杜伟,丁雄,陈晓光,简华刚,龚建平,刘长安.靶向重组腺病毒载体逆转肝癌细胞多药耐药的实验研究[J].陆军军医大学学报(原第三军医大学学报),2009,31(15):1462-1466. DOI:
MEI Ying,DU Wei,DING Xiong,CHEN Xiao-guang,JIAN Hua-gang,GONG Jian-ping,LIU Chang-an.Experimental study of the targeting recombinant adenoviral vector in reversing the mdr hepatocarcinoma cells[J].J Amry Med Univ (J Third Mil Med Univ),2009,31(15):1462-1466. DOI:
目的 探讨携带多药耐药基因1(multidrug resistance gene 1,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; Rhodamine 123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(P0.05),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 efficacy and the possible action mechanisms of the recombinant adenovirus vector carrying antisense multidrug resistance gene 1 (mdr1) targeting the reverse of α-fetoprotein (AFP+) positive MDR of HepG2R cells. Methods After transfection of the recombinant adenoviral vector Adeno-EGFP carrying AFP promoter and EGFP gene into LO2
a normal human liver cell line (AFP-)
HeLa (AFP-)
a human cervical cell line
and HepG2 (AFP+)
the transcription level and the EGFP protein expression were detected by Northern blot and fluorescence microscopy. After the transfection of the recombinant adenovirus vector Adeno-asmdr carrying AFP promoter and antisense mdr1 into HepG2R cells and subcutaneous transplanted tumor carrying HepG2R in nude mice
the reverse activity of HepG2R cells was detected in vivo and in vitro. Results The results showed that the EGFP gene and protein were markedly transcribed and expressed in HepG2 cells
however
they were very weak in LO2 or HeLa cells. The results also showed a good transcription ability and AFP-targeting specificity of the recombinant adenoviral vector. RT-PCR showed that the mdr1 was markedly down-regulated at 7 d after transfection. Rhodamine 123 efflux tests showed that the retention of the fluorescence reagent was markedly increased in HepG2R cells at 48 h after transfection. The tumor volume following injection with Adeno-asmdr plus ADM group did not increase
but increased significantly in the PBS and ADM groups (P0.05). A high rate of apoptosis of cells was detected by pathological assay in the treatment group with ADM plus Adeno-asmdr. Conclusion The recombinant adenoviral expression vector could express the specific target gene in AFP producing hepatocarcinoma cell and block the mRNA expression of mdr1
down-regulate p-gp170
and reverse MDR. Combined with chemotherapeutic agents
the adeno-asmdr could partly reverse the mdr1 gene expression in vivo. It has a potential value for clinical application.
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