eola1下调表达对ECV304细胞增殖的影响

目的设计和构建人类新基因人内皮高表达脂多糖相关因子1(endothelium-overexpressed lipopolysaccharide-associated factor1, eola1)特异性的小干扰RNA表达载体sieola1,建立eola1下调表达模型,观察eola1下调表达对细胞增殖的影响.方法设计靶点特异性的寡核苷酸,连接到经BamHⅠ-HindⅢ酶切线性化的pSlincer3.1/H1质粒上;转染重组质粒到ECV304细胞,通过定量RT-PCR实验检测靶基因的抑制情况;对转染sieola1质粒的细胞进行细胞计数,并应用流式细胞仪观察细胞周期的变化.结果构建的小RNA干扰质粒sieola1转染ECV304细胞能够抑制靶基因eola1的表达;细胞计数和流式细胞仪检测结果显示eola1表达抑制后,细胞生长周期延长.结论成功构建了针对eola1基因的siRNA质粒;细胞计数和流式细胞仪检测结果初步确认eola1具有抑制ECV304细胞增殖的作用.

Effects of down-regulated expression of eola1 on the proliferation of ECV304 cells

Objective To design and construct H1 promoter-based small interfering RNA (siRNA) expression vectors and to detect the effects of down-regulated expression of endothelium overexpression LPS associated factor 1 (eola1) on the proliferation of ECV304 cells. Methods DNA oligonucleotides targeting eola1 were synthesized and inserted into BamHI-HindIII linearized pSinencer3.1/H1 plasmid. The empty vector pSilencer3.1/H1 and recombinant vector sieola1 were transfected into the ECV304 cells. The expression level of eola1 of transfected cell strains was measured by RT-PCR. The cell growth of transfected cell strains was observed by cell count and flow cytometry. Results The vector-base RNAi could knockdown eola1 gene expression specifically. The down-expression of eola1 accelerated the proliferation of ECV304 cells. Conclusion Vector-based siRNA expression plasmids could down-regulate the expression of targeting gene - eola1, which accelerates the proliferation of ECV304 cells.