摘要
目的 探讨WT1基因沉默对人类白血病细胞血管内皮生长因子(VEGF)表达的影响.方法 将已构建的WT1小发夹RNA(shRNA)质粒载体转染入K562细胞,流式细胞术检测转染效率并对转染细胞进行分选,实时荧光定量PCR检测细胞转染前后WT1及VEGF基因mRNA的表达变化,用ELISA检测转染前后细胞培养液中VEGF浓度的变化.结果 转染48h,WT1shRNA质粒转入K562细胞,转染效率为(65±4.5)%,通过流式细胞术分选,可以得到纯度达98%以上的WT1shRNA阳性细胞,转染后的K562细胞WT1及VEGF基因mRNA表达较转染前及对照组均明显降低(P＜0.05),转染后48、72h细胞培养液中VEGF的含量较对照组明显降低(P＜0.05).结论 WT1干扰质粒在体外可以抑制白血病细胞株VEGF的表达,提示WT1基因可能通过上调VEGF基因的表达而参与白血病的血管新生.
Objective To investigate the effect to the expression of VEGF in human leukemic cell line by WT1gene silencing. Methods K562 cells were transfected with WT1shRNA Plasmid.Transfection efficiency was detected by flow cytometIy,and the succeed transfered cells Were sorted.WT1 and VEGF mRNA variation were measured by fluorescence RT-PCR. VEGF concentrations in the cell growth medium were detected by ELISA.Results 48 hours after transfection,the transfection efficiency was 65％±4.5％.WT1shRNA positive cells were sorted by flow cytometry.Both the mRNA level of WT1 and VEGF and VEGF concentrations in cell growth medium were significantly decreased than the control group. Conclusion WT1 gene silence can down-regulate the expression of VEGF in K562 cells which suggest that WT1 gene may take part in leukemia angiogenesis through up-regulation of VEGF.
出处
《肿瘤研究与临床》
CAS
2008年第3期152-154,157,共4页
Cancer Research and Clinic
基金
山西省自然基金资助项目(20051102)
