腺病毒介导PDCD5基因转移促进依托泊甙诱导的K562细胞凋亡_医学论文.docVIP

腺病毒介导PDCD5基因转移促进依托泊甙诱导的K562细胞凋亡_医学论文 腺病毒介导PDCD5基因转移促进依托泊甙诱导的K562细胞凋亡_医学论文 作者：阮国瑞， 陈珊珊， 常艳， 李金兰， 秦亚溱， 李玲娣， 郝乐， 付家瑜， 刘艳荣， 黄晓军【摘要】 本研究观察程序性细胞死亡5基因（programmed cell death 5， PDCD5）重组腺病毒转染K562细胞后对化疗药物依托泊甙的增敏作用。 利用AdMaxTM腺病毒载体包装系统，通过同源重组方法构建Ad重组腺病毒及对照腺病毒Ad及Ad；用不同感染复数将Ad、Ad或Ad转染人白血病细胞系，实时定量PCR检测PDCD5 mRNA的相对表达水平；利用MTT法及Annexin双染色流式细胞术观察依托泊甙对转染后K562细胞增殖与凋亡的影响。结果表明： Ad腺病毒对白血病细胞系K562、Jurkat及CEM的转染效率可达60%-86%。Ad重组腺病毒能梯度增加K562细胞PDCD5 mRNA的相对表达水平，腺病毒介导的PDCD5基因转移促进依托泊甙诱导的K562细胞凋亡。结论： PDCD5重组腺病毒可能成为化疗药物的增敏剂。 【关键词】 腺病毒 Adenovirus Abstract This study was purposed to investigate the effect of adenovirusmediated transfer of PDCD5 gene on apoptosis of K562 cells induced by etoposide. Recombinant adenovirus PDCD5 (AdPDCD5), control vectors Adnull and AdeGFP were constructed by AdMaxTM vector system respectively. After K562 cells were transfected by AdPDCD5, Adnull or AdeGFP with different multiplicity of infection (MOI), the expression level of the PDCD5 gene was examined by RQRTPCR assay. The effects of etoposide in combination with AdPDCD5 on the proliferation and apoptosis of K562 cells were measured by using MTT assay and flow cytometry with AnnexinVFITC/PI dual labeling technique, respectively. The results showed that the transfection efficiencies of AdeGFP in K562, Jurkat and CEM cells ranged from 60% to 86%. Expression level of PDCD5 gene in K562 cells was evidently increased following transfection with AdPDCD5. The AdPDCD5 synergistically enhanced the apoptotic percentage of K562 cells induced by VP16, as compared with that of Adnull+VP16 and VP16 alone respectively. It is concluded that AdPDCD5 may be a potential agent for enhancing the chemotherapy effect. Key words adenoviruses； PDCD5； etoposide K562 cell line； apoptosis； gene transfer TF细胞凋亡相关基因（TF，TFAR19）是1999年发现的一种新的凋亡调控基因（GenBank登记号为AF014955）［1］，国际人类基因命名委员会将其命名为PDCD5 (programmed cell death 5)，将含PDCD5 cDNA 的真核表达载体转染白血病细胞系TF等细胞后，可加速这些细胞的凋亡。已发现PDCD5在白血病细胞系Jurkat凋亡过程中发挥作用［2］，人重组PDCD5蛋白对白血病细胞系HL具有促凋亡作用［3