Kang Yu, Zhang Xiao-Yan, Jiang Wei, Wu Chao-Qun, Chen Chun-Mei, Gu Jian-Ren, Zheng Yu-Fang, Xu Cong-Jian
Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.
Cell Biol Int. 2009 Apr;33(4):509-15. doi: 10.1016/j.cellbi.2009.01.017.
Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 +/- 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was 10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.
基因导向酶前药疗法（GDEPT）是一种为选择性靶向癌细胞而开发的策略。然而，由于其基因转移效率低下，临床获益有限。为克服这一障碍，我们利用了猪尾巴（PB）转座子，这是一种天然的非病毒基因载体，可在包括人类细胞在内的脊椎动物细胞中诱导稳定的染色体整合和持续的基因表达。为确定该载体是否也能介导卵巢癌细胞中的稳定基因表达，我们构建了一个同时表达单纯疱疹病毒胸苷激酶（HSV-tk）基因和单体红色荧光蛋白（mRFP1）报告基因的PB转座子系统。用FuGENE HD试剂将重组质粒pPB/TK转染至卵巢腺癌细胞SKOV3中，通过流式细胞术和共聚焦显微镜检测mRFP1阳性细胞的百分比来评估转染效率。通过RT-PCR和蛋白质印迹法证实转染细胞中HSV-tk的特异性表达。采用甲基噻唑基四氮唑（MTT）法测定转染细胞对前药更昔洛韦（GCV）的敏感性。与转染pORF-HSVtk的细胞中未检测到可测量的mRFP1表达相比，用pPB/TK转染的细胞中共有56.4±8.4%为mRFP1阳性。pPB/TK转染细胞中HSV-tk的表达水平比pORF-HSVtk转染细胞高10倍。结果表明，pPB/TK转染以剂量依赖的方式增加了细胞对GCV的敏感性。我们的数据表明，PB转座子系统可提高GDEPT在卵巢癌中的抗肿瘤效率。
