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目的:研究siRNA抑制Skp2基因在食管癌细胞中表达,探讨Skp2在肿瘤发生过程中的作用。方法:在Skp2基因编码区选择4个RNAi作用位点,体外合成前体DNA链,复性后连入逆转录病毒载体pSUPER-retro(pRS),转染包装细胞PT67,收集含病毒颗粒的培养上清感染食管癌Eca-109细胞,经嘌呤霉素筛选并扩大培养后形成稳定克隆。流式细胞仪检测细胞凋亡情况,RT-PCR和免疫印迹法检测Eca-109细胞内Skp2 mRNA、p27和Bcl-2表达。结果:与正常细胞和空载细胞相比,筛选得到的2个克隆细胞Skp2 mRNA表达水平下降(P<0.01),p27表达增高(P<0.05),Bcl-2表达降低(P<0.05),细胞凋亡增加(P<0.05)。结论:Skp2 siRNA促进食管癌细胞凋亡与p27上调有关。
Objective: To study the effect of siRNA on the expression of Skp2 in esophageal cancer cells and to explore the role of Skp2 in tumorigenesis. METHODS: Four RNAi sites were selected in the Skp2 gene coding region. The DNA strand was synthesized in vitro and inserted into the retroviral vector pSUPER-retro (pRS) after refolding. The packaging cells were transfected with PT67 to collect virus particles The supernatant was infected with esophageal cancer Eca-109 cells, which were screened with puromycin and expanded to form stable clones. Cell apoptosis was detected by flow cytometry. The expression of Skp2 mRNA, p27 and Bcl-2 in Eca-109 cells was detected by RT-PCR and Western blot. Results: Compared with normal cells and empty cells, the expression of Skp2 mRNA decreased (P <0.01), the expression of p27 increased (P <0.05) and the expression of Bcl-2 decreased Apoptosis increased (P <0.05). Conclusion: Skp2 siRNA can promote the apoptosis of esophageal cancer cells and the up-regulation of p27.