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目的探讨靶向Lin28B基因沉默对细胞增殖的影响。方法设计Lin28B及GFP短发夹核糖核酸(shRNA)序列,装入Tet-on-pLKO-puro质粒,进行慢病毒包装,感染胰腺癌细胞Panc1,嘌呤霉素筛选并培养稳定干扰细胞株。实验组为Panc1-Tet-on-pLKO-sh-Lin28B;对照组为Panc1-Tet-on-pLKO-sh-GFP。筛选强力霉素最佳诱导浓度并诱导干扰序列表达,Western blot检测干扰效率,细胞增殖与活性实验CCK8检测沉默Lin28B对Panc1细胞增殖的影响。结果构建的Tet-on-pLKO-sh-Lin28B慢病毒可显著抑制Panc1细胞内Lin28B的表达。与对照组Panc1-Tet-onpLKO-sh-GFP相比,实验组Panc1-Tet-on-pLKO-sh-Lin28B的增殖速度减缓(P<0.05)。结论成功筛选出沉默Lin28B的稳定干扰细胞株,沉默Lin28B对胰腺癌细胞的增殖有抑制作用,为进一步研究Lin28B基因的功能或基因治疗提供有用技术。
Objective To investigate the effect of targeted silencing of Lin28B gene on cell proliferation. Methods The short hairpin RNA (shRNA) sequences of Lin28B and GFP were designed and inserted into Tet-on-pLKO-puro plasmid for lentivirus packaging. Panc1 cells were infected with puromycin and stable interfering cell lines were obtained. The experimental group was Panc1-Tet-on-pLKO-sh-Lin28B; the control group was Panc1-Tet-on-pLKO-sh-GFP. The optimal inducing concentration of doxycycline was screened and the expression of interference sequence was induced. The interference efficiency was detected by Western blot. The effect of silencing Lin28B on the proliferation of Panc1 cells was detected by CCK8 assay. Results The constructed Tet-on-pLKO-sh-Lin28B lentivirus significantly inhibited the expression of Lin28B in Panc1 cells. Compared with the control group Panc1-Tet-onpLKO-sh-GFP, the experimental group Panc1-Tet-on-pLKO-sh-Lin28B slowed down (P <0.05). Conclusion The stable interfering cell lines with silenced Lin28B were screened successfully. Silencing of Lin28B could inhibit the proliferation of pancreatic cancer cells and provide a useful technique for further study on the function and gene therapy of Lin28B.