中文摘要：

目的：构建抑癌基因膀胱癌相关蛋白（BLCAP）基因高效真核siRNA载体,转染人宫颈癌细胞系HeLa,构建BLCAP基因沉默细胞系,以观察靶向siRNA对BLCAP基因的表达抑制作用。方法：设计并重组靶向siRNA各3条,分别命名为pRNA-U6.1-B1,B2,B3-siRNA,重组体经脂质体转染细胞,通过荧光显微镜观察细胞内质粒表达情况,并采用RT-PCR和Western blot方法检测转染前后BLCAP基因mRNA及蛋白的表达,验证RNAi作用的特异性及时效性。结果：同对照组相比,各BLCAP siRNA均可引起靶基因表达水平的显著性下降（P〈0.05）,尤以pRNA-U6.1-B2-siRNA的作用效果最强（P〈0.01）;而空脂质体组及阴性对照siRNA组的靶基因表达水平则无明显变化。将pRNA-U6.1-B2-siRNA转染细胞后12,24,48h均可观察到靶基因表达的显著性降低（P〈0.05）,其中在24h后达到最强抑制效果。结论：不同序列的siRNA,对靶基因的干扰效率不同;BLCAP基因siR-NA能够特异、高效性的抑制靶基因的表达;抑制作用在24h达到高峰,并可维持到转染后48h。试验结果为进一步研究采用RNA干扰技术进行宫颈癌的生物治疗奠定了基础。

英文摘要：

Objective： To construct cell lines with silenced bladder cancer-associated protein（BLCAP） with novel designed small interfering RNAs（siRNAs）.Methods： Three specific siRNAs targeting BLCAP gene were designed and cloned separately,and were named as pRNA-U6.1-B1,B2,B3-siRNAs.The inhibitory effect and time-efficiency of each siRNA were investigated,and the expression levels of mRNA and protein of the target genes were detected with RT-PCR and Western blot at 12,24 and 48 h after transfection with the siRNAs.Results： Each siRNA led to a downregulation of the expression of target genes（P0.05）,but the degree of downregulation was different.No distinct changes were found in the group transfected with liposomal transfection reagent without siRNAs.The results indicated that pRNA-U6.1-B2-siRNA was the most effective siRNA.Transfection with pRNA-U6.1-B2-siRNA induced a marked decrease of corresponding BLCAP mRNA and protein levels at different time points（P0.05） when compared with the control group.Conclusion： Inhibitory effect varies with siRNAs targeting different sequences of the same gene.Transfection with either siRNAs induces a specific and efficient downregulation of BLCAP gene in HeLa cells positive for HPV16 that lasts for 48 hours.