Targeted killing effect of lentivirus-mediated CD/TK suicide genes on colorectal carcinoma cells
- Author:
- No author available
- Journal Title:
- WORLD CHINESE JOURNAL OF DIGESTOLOGY
- Issue:
- 17
- DOI:
- 10.3969/j.issn.1009-3079.2006.17.006
- Key Word:
- 大肠癌;KDR启动子;自杀基因;慢病毒
Abstract： 目的:研究慢病毒介导的KDR启动子驱动CD/TK双自杀基因系统对大肠肿瘤细胞选择性杀伤作用.方法:构建的重组质粒pLenti6/V5-D-KDR-CDglyTK及阳性对照质粒pLenti6/V5-D-GFP在lipofectamine 2000介导下转染293FT细胞,包装扩增后获得病毒颗粒,体外感染表达KDR的SW620细胞株和不表达KDR的LS174T细胞株,荧光显微镜下计数确定阳性对照质粒的感染效率并以RT-PCR方法检测转基因细胞CDglyTK的表达,然后给予不同浓度的前药5-FC及GCV处理,观察该体系对不同细胞株的杀伤效应及其旁观者效应.结果:重组体对各细胞株的感染率相似,其感染率随慢病毒滴度的增高而递增.RT-PCR方法检测发现:除LS174T细胞外,SW620细胞有目的基因CDglyTK的表达.对于转基因的SW620细胞,单用GCV(100mg/L)时,其存活率为32.34%±2.42%.单用5-FC(2.0 g/L)时,存活率为30.56%±2.14%.而联合应用两者时,SW620细胞存活率为5.36%±1.55%,LS174T细胞存活率为95.48%±1.70%.SW620细胞对前药具有较高的敏感性,SW620与LS174T细胞株对前药敏感性有显著性差异(P＜0.001),双自杀基因的疗效优于任一单自杀基因的疗效(P＜0.001).当转基因细胞比率为40%时,加入前药GCV和5-FC,SW620细胞存活率为11.42%±2.66%,而LS174T细胞存活率为99.54%±2.61%,二者比较差异有显著性意义(P＜0.001),该体系旁观者效应明显.结论:慢病毒作为载体有较高的转染效率,KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的大肠肿瘤细胞.
- [1]杨文宇,黄宗海.自杀基因治疗恶性肿瘤的载体效率及基因表达[J].2004,(1).
- [2]李振宇,徐开林,潘秀英.慢病毒载体构建及结构优化[J].2002,(5).
- [3]Morris JC,Bukovsky AA,Kurre P,etc.Lentivirus-mediated gene transfer into hematopoietic repopulating cells in baboons.[J].2002,9(21).
- [4]Ross CA,de Almeida LP,Aebischer P,etc.Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length.[J].2002,22(9).
- [5]S I, Gilles,S, Romain,P, Casellas,etc.Mutation analysis in the coding sequence of thymidine kinase 1 in breast and colorectal cancer.[J].1900,18.
- [6]Lo Bianco C,Ridet JL,Schneider BL,etc.alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease.[J].2002,99(16).
- [7]Trottier Y,Perrin V,Aebischer P,etc.Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum.[J].2003,12(21).
- [8]Y, Harada,Y, Ogata,K, Shirouzu.Expression of vascular endothelial growth factor and its receptor KDR (kinase domain-containing receptor)/Flk-1 (fetal liver kinase-1) as prognostic factors in human colorectal cancer.[J].2001,6(5).
- [9]Day D,Naylor S,Binley K,etc.Hypoxia-mediated tumour targeting.[J].2003,10(7).
- [10]McManus MT,Scott ML,Kwiatkowski AV,etc.A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.[J].2003,33(3).
- [11]Park F,Kay MA,Ohashi K.The effect of age on hepatic gene transfer with self-inactivating lentiviral vectors in vivo.[J].2003,8(2).
- [12]Verma IM,Ikawa M,Pfeifer A,etc.Transgenesis by lentiviral vectors: lack of gene silencing in mammalian embryonic stem cells and preimplantation embryos.[J].2002,99(4).
- [13]Weppert M,Kessler B,Ludwig H,etc.Efficient transgenesis in farm animals by lentiviral vectors[J].2003,4(11).
- [14]Dai W,Langham MR Jr,Copeland EM 3rd,etc.Expression of VEGF receptors in cocultured neuroblastoma cells.[J].2004,119(1).
- [15]Linderholm B,Emdin S,Nielsen NH,etc.Tumor specific VEGF-A and VEGFR2/KDR protein are co-expressed in breast cancer.[J].2003,82(3).
- [16]DeFatta RJ,De Benedetti A,Li Y.Selective killing of cancer cells based on translational control of a suicide gene.[J].2002,9(7).
- [17]Qi L,Glode LM,Robinson WA,etc.Migration and invasion of human prostate cancer cells is related to expression of VEGF and its receptors.[J].2003,23(5A).
- [18]Zakhartchenko V,Wolf E,Hofmann A,etc.Generation of transgenic cattle by lentiviral gene transfer into oocytes[J].2004,71(2).
- [19]Ozawa K,Hanazono Y,Terao K.Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy.[J].2001,19(1).
- [20]Nguyen TH,Morel P,Majno P,etc.Highly efficient lentiviral vector-mediated transduction of nondividing, fully reimplantable primary hepatocytes.[J].2002,6(2).