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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。
OBJECTIVE: To construct high efficient eukaryotic siRNA vector of bladder cancer related protein (BLCAP) gene of tumor suppressor gene and transfect it into HeLa cell line of human cervical cancer to construct BLCAP gene silencing cell line to observe the inhibitory effect of targeted siRNA on BLCAP gene expression. Methods: Three siRNAs were designed and recombined respectively and named as pRNA-U6.1-B1, B2, B3-siRNA. The recombinant plasmids were transfected into cells by lipofectamine. The expression of plasmids was observed by fluorescence microscopy The mRNA and protein expression of BLCAP before and after transfection were detected by RT-PCR and Western blot to verify the specificity and timeliness of RNAi. Results: Compared with the control group, all BLCAP siRNAs significantly decreased the expression of target genes (P <0.05), especially pRNA-U6.1-B2-siRNA had the strongest effect (P <0.01). However, there was no significant change in target gene expression in empty liposome group and negative control siRNA group. After transfected with pRNA-U6.1-B2-siRNA for 12, 24 and 48h, the expression of target gene was significantly decreased (P <0.05), and the strongest inhibitory effect was achieved after 24h. CONCLUSIONS: siRNAs of different sequences have different interference efficiency on target genes. The siR-NA of BLCAP gene can specifically and efficiently inhibit the target gene expression. The inhibitory effect peaked at 24h and remained at 48h after transfection. The results of the study laid the foundation for the further study of the biological treatment of cervical cancer by using RNA interference technology.