摘要
目的针对内源性Ku80基因序列体外构建短干扰RNA(siRNA)线性DNA转录载体,观察其在细胞内产生的siRNA对该基因表达的影响。方法构建的线性DNA转录载体经脂质体导入宫颈癌HeLa细胞内,转录产生21 bp的短双链siRNA,并用免疫印迹法分析Ku80基因在蛋白水平表达的情况。结果体外成功构建出siRNA线性DNA转录载体LS1/LAS1和LS2/LAS2;载体分别导入HeLa细胞内生成相应siRNA;与空白对照组比较,实验组均能特异性抑制Ku80基因的表达;其抑制效果和时间点与所选基因靶位相关。结论由线性DNA载体转录生成的siRNA在细胞内特异性抑制内源性基因表达,为Ku80用于肿瘤靶向基因治疗提供了一个新的实验依据。
Objective To develop a linear DNA vector approach targeting the Ku80 gene sequence and investigate the inhibitory effect of short interfering RNA （siRNA） on Ku80 expression. Methods Linear DNA vectors with human U6 RNA-based polymerase Ⅲ promoter and modified terminator for precise siRNA production were amplified by PCR in vitro, transfected into HeLa cells, and the siRNA to induce RNA interfering was synthesized. The protein expression level of intracellular Ku80 was detected by Western-blotting. Results In vivo, linear DNA vectors （LS1/LAS1 and LS2/LAS2） were successfully constructed and expressed siRNA in HeLa cells, by which the posttranscriptional expression of Ku80 gene was specially silenced compared with the control group. The inhibitory effect and time slice were related to the targeted gene site. Conclusion siRNA specifically suppressed Ku80 gene expression, which provided a new and potential tumor gene therapy for Ku80 as a candidate target.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2006年第3期400-402,410,共4页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
作者简介
熊华，男，1978年生，住院医师，医学硕士
