MDR1启动子调控的双自杀基因靶向杀伤耐药胶质瘤细胞的研究

山东大学学报(医学版)

MDR1启动子调控的双自杀基因靶向杀伤耐药胶质瘤细胞的研究

王波¹，王志云²，王成伟¹，王志刚¹，张源¹，张庆林¹

1. 山东大学第二医院神经外科，济南 250033；2. 潍坊市人民医院，山东潍坊 261041

收稿日期:2007-05-30 修回日期:1900-01-01 出版日期:2008-01-16 发布日期:2008-01-16
通讯作者: 王成伟

Targeted killing effects of double suicide genes controlled by the MDR1 promoter on C6/ADR cells

WANG Bo¹，WANG Zhi-yun²，WANG Cheng-wei¹，WANG Zhi-gang¹，ZHANG Yuan¹，ZHANG Qing-lin¹

1. Department of Neurosurgery, Second Hospital of Shandong University;2. Weifang People′s Hospital, Weifang 261041, Shandong, China

Received:2007-05-30 Revised:1900-01-01 Online:2008-01-16 Published:2008-01-16
Contact: WANG Cheng-wei

摘要/Abstract

摘要： 目的探讨多药耐药基因（MDR1）启动子调控的CD、TK双自杀基因系统并加前体药物丙氧鸟苷及5氟胞嘧啶（pcDNA3.MDR1P.CDTK/GCV+5-FC）对耐药胶质瘤细胞（C6/ADR细胞）的靶向杀伤作用。方法利用脂质体介导法将含有MDR1启动子调控的CD、TK双自杀基因的真核表达载体PcDNA3.MDR1P.CD.TK转染入C6/ADR细胞（C6/ADR/CDTK），利用PCR鉴定CD、TK基因的整合，利用RT-PCR鉴定CD、TK基因的表达；然后以转染了质粒pcDNA3.MDR1P.CDTK的C6细胞（C6/CDTK）和正常C6/ADR细胞作为对照，分别加前体药物，利用生长曲线、流式细胞仪、平板克隆形成等方法研究双自杀基因对耐药胶质瘤细胞生长、细胞周期及增殖的影响。结果PCR结果显示，CD、TK基因均整合入C6和C6/ADR细胞中；RT-PCR结果显示，C6/ADR/CDTK细胞中CD、TK基因有特异性表达，而C6/CDTK细胞无特异性表达。应用前体药物后，C6/ADR/CDTK细胞增殖明显受抑；C6/ADR、C6/CDTK、C6/ADR/CDTK细胞经流式细胞仪检测G1期的细胞比例分别为32.68%、47.57%、99.93%（P＜0.05，其平板克隆形成率分别为(96.7±2.1)%、(86.7±1.9)%、(16.7±0.9)%（P＜0.05）。结论pcDNA3.MDR1P.CDTK/GCV+5-FC系统对C6/ADR具有较强的靶向杀伤作用。

Abstract: Objective To investigate the targeted killing effects of a double suicide gene system controlled by the MDR1 promoter with the prodrug GCV+5-FC on C6/ADR cells. Methods The recombinant vector pcDNA3.MDR1P.CD.TK containing CD and TK double suicide genes controlled by the MDR1 promoter was transfected into C6/ADR cells by using liposome. PCR and RT-PCR were used to identify the integration and expressions of the CD and TK genes. This recombinant vectortransfected C6 cells (C6/CDTK) and normal C6/ADR cells were set as controls. After adding the prodrug, the effects of double suicide genes on cell growth, cell cycle and proliferation were determined by cell growth curve, flow cytometry (FCM) and plate colony formation assay. ResultsPCR proved double suicide genes were integrated into C6/ADR and C6 cells. RT-PCR revealed that the CD and TK genes were expressed in C6/ADR/CDTK cells C6/ADR/CDTK cells was inhibited. G1 phase proportion was significantly higher in C6/ADR/CDTK cells (99.93%) than in C6/ADR cells and C6/CDTK cells (32.68% and 47.57%, P<0.05); the formation rate was significantly lower in C6/ADR/CDTK cells ［(16.7±0.9)%］ than in C6/ADR cells and C6/CDTK cells ［(96.7±2.1)% and (86.7±1.9)%, P<0.05］. ConclusionThe pcDNA3.MDR1P.CDTK/GCV+5FC system has targeted killing effects in C6/ADR cells.

Key words: Multidrug resistance gene, Promoter, Transfection, Glioma, Gene therapy

中图分类号:

R739.41

王波,王志云,王成伟,王志刚,张源,张庆林 . MDR1启动子调控的双自杀基因靶向杀伤耐药胶质瘤细胞的研究[J]. 山东大学学报(医学版), 2008, 46(1): 15-18.

WANG Bo,WANG Zhi-yun,WANG Cheng-wei,WANG Zhi-gang,ZHANG Yuan,ZHANG Qing-lin. Targeted killing effects of double suicide genes controlled by the MDR1 promoter on C6/ADR cells[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2008, 46(1): 15-18.

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