辐射诱导分子lnc-RI稳定敲低细胞系建立及其功能

Establishment of stable knock-down cell lines and function study of lnc-RI, a radiation-induced gene

制备lnc-RI干涉表达的慢病毒颗粒,建立lnc-RI稳定下调的He La细胞系,探索lnc-RI对细胞辐射敏感性的作用。首先将lnc-RI特异性干涉序列插入干涉表达载体p GLV2中,并制备慢病毒颗粒;然后,将慢病毒颗粒感染He La细胞,建立lnc-RI稳定下调的细胞系,并通过RT-PCR检测不同干涉序列对lnc-RI表达的干涉效果;最后,通过平板克隆形成实验分析lnc-RI表达下调对He La细胞放射敏感性的影响。结果显示,成功制备了携带lnc-RI干涉序列的慢病毒颗粒,并成功感染He La细胞,通过RT-PCR实验鉴定获得两株lnc-RI稳定敲低细胞系。克隆形成实验发现,与对照相比,lnc-RI敲低的He La细胞系的放射敏感性增加。因此,初步证实lnc-RI表达下调增加He La细胞放射敏感性。

To prepare the lentivirus granules interfering lnc-RI and establish HeLa cell lines consistently down-regulating lnc-RI, as well as discover its effects on radiation damage, the lentivirus granules bearing various interfering sequences were prepared by lnc-Rl carrying specific interfering sequences in to interfering plasmid pGLV2. The estable cell lines were established after infecting HeLa cells with lentivirus granules. RT-PCR was employed to determine the inhibitory effects of various interfering sequences on lnc-RI expression. DNA damage of the established estable cell lines were induced by ionizing radiation sensitivity of the established cell lines which were determined by colony formation assay. The results showed that the lentivirus granules bearing various interfering sequences were successfully prepared. The estable HeLa cell lines infected by lentivirus were obtained and lnc-RI expression levels was down-regulated. The results of colony formation assay showed that radiation sensitivity of the estable cell lines increased when treated with each dose gamma ray irradiation.