Xu Wen-lin, Shen Hui-ling, Wu Zhao-yang, Tang Hua-rong, Wang Fa-chun
Department of Hematology, The Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu 212002, P.R.China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2006 Feb;23(1):37-42.
To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562.
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. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA 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.
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 transfected with empty vector) cells. The gene expression profiles 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.
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.
探讨抗血管内皮生长因子（VEGF）发夹状核酶基因对白血病细胞株K562基因表达谱的潜在影响。
采用脂质体介导法，将含抗VEGF发夹状核酶基因的重组真核表达质粒（pcDNA3-RZ）及作为对照的非重组载体转染至K562细胞。用G418筛选阳性克隆。通过聚合酶链反应（PCR）确认K562细胞中的核酶基因。采用荧光实时逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测白血病细胞中VEGF信使核糖核酸（mRNA）和蛋白的表达。利用cDNA微阵列技术，探讨白血病细胞中VEGF基因表达降低时基因表达谱的变化。通过半定量RT-PCR验证增殖细胞核抗原（PCNA）和凝溶胶蛋白（GSN）基因的表达。
将pcDNA3-RZ和pcDNA3转染至人白血病细胞株K562，并通过G418筛选出阳性克隆。通过PCR确认K562细胞中核酶基因的稳定表达。与K562或K562-PC（转染空载体的K562细胞）细胞相比，K562-RZ细胞中VEGF mRNA和蛋白水平显著降低。将抗VEGF发夹状核酶基因转染至K562细胞后，基因表达谱发生改变。在微阵列上的4096个基因克隆中，检测到191个（4.86%）基因有明显变化，其中104个下调，87个上调，这些基因在功能上与细胞周期进程、基因复制、代谢、细胞凋亡、细胞信号转导和癌基因等相关。通过RT-PCR检测到K562/RZ细胞中GSN基因表达增加，PCNA基因表达降低。
导入抗VEGF发夹状核酶基因下调VEGF基因可改变K562细胞的基因表达谱，导致K562/RZ细胞的生长、分化和凋亡发生改变。
