摘要
目的探讨血管内皮细胞生长因子(vascular endothelial growthfactor,VEGF)基因VEGF表达下调对白血病K562细胞株基因表达谱的影响。方法采用脂质体介导的方法将抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转染白血病细胞株K562、G418抗性筛选获得阳性克隆;抽提基因组DNA,用PCR方法验证核酶基因已转入K562细胞;荧光定量PCR和免疫印迹反应检测白血病细胞中VEGFmRNA和蛋白表达量的改变;应用cDNA微阵列技术检测VEGF基因表达下调对白血病K562细胞株基因表达谱的影响。并用逆转录PCR验证PCNA、GSN基因的表达改变。结果抗VEGF发夹状核酶基因真核表达载体pcDNA-RZ转入白血病细胞株K562、G418筛选两周获得阳性克隆,PCR检测证实核酶基因整合入白血病细胞基因组DNA;与K562及K562/PC细胞(转染空质粒的K562细胞)相比,转染VEGF核酶基因的K562/RZ细胞VEGFmRNA和蛋白的表达量明显降低,芯片中共有表达差异的基因191条,包括周期相关基因、细胞凋亡相关基因、癌基因以及细胞信号和传递蛋白等基因,其中104条表达下调,87条表达上调。逆转录PCR证实GSN基因表达上调,PCNA基因表达下调。结论VEGF基因表达下调能引起白血病K562细胞株基因表达谱的改变,这些基因的改变可能对白血病细胞增殖、分化和凋亡等生物学行为产生了一定的影响。
Objective To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemja cell line K562. Methods The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid （pcDNA3-RZ） containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Rihazyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR（RT-PCR） and Westem blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells, eDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR. Results The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC （K562 cell transfeeted with empty vector） cells. The gene expression pmfdes were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 （4.86%） genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR. Conclusion Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2006年第1期37-42,共6页
Chinese Journal of Medical Genetics
