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目的:利用RNA干扰技术构建并筛选出对Nanog基因有抑制作用的重组质粒pshRNA-Nanog,转染入胃癌细胞SGC-7901中,检测其对Nanog表达的影响及其对胃癌细胞SGC-7901的增殖、周期、凋亡以及迁移的影响。方法:构建出针对人Nanog基因构建出的干扰质粒pshRNA-Nanog,在脂质体的介导下将其转染胃癌细胞SGC-7901,通过荧光显微镜,RT-PCR和Western blot法检测其表达,筛选出抑制率最高的一组;将筛选出的转染效率最高的一组pshRNA-Nanog重组质粒转染胃癌细胞SGC-7901中,通过CCK-8检测其胃癌细胞SGC-7901增殖情况,流式细胞仪分析SGC-7901周期变化和凋亡情况,Transwell迁移实验验证Nanog对SGC-7901的迁移能力以及侵袭实验验证Nanog对SGC-7901侵袭能力的影响。结果:成功构建并筛选出了对胃癌细胞SGC-7901干扰效果最明显的一组重组质粒;细胞的增殖受到抑制,胃癌细胞侵袭能力及迁移能力减弱,细胞周期停滞在S期,细胞的凋亡明显增加。结论:Nanog基因与胃癌细胞的增殖能力、周期及凋亡、迁移和侵袭能力有密切的关系。
OBJECTIVE: To construct and screen a recombinant plasmid pshRNA-Nanog with inhibition of Nanog gene by RNA interference and transfect it into SGC-7901 gastric cancer cell line SGC-7901 to detect its effect on the expression of Nanog and its effect on the proliferation of gastric cancer cell line SGC-7901 , Cycle, apoptosis and migration. METHODS: The interference plasmid pshRNA-Nanog constructed from human Nanog gene was constructed and transfected into human gastric cancer cell SGC-7901 by lipofectamine. The expression of pshRNA-Nanog was detected by fluorescence microscope, RT-PCR and Western blot. A group of the highest transfection efficiency of pshRNA-Nanog recombinant plasmids was transfected into SGC-7901 gastric cancer cell line SGC-7901 to detect the proliferation of gastric cancer cell line SGC-7901 by flow cytometry Cell cycle analysis and SGC-7901 cycle changes and apoptosis, Transwell migration experiments to verify the migration of SGC-7901 Nanog migration and invasion experiments to verify the invasion of SGC-7901 Nanog impact. Results: A group of recombinant plasmids were successfully constructed and screened for the most obvious interference effect on gastric cancer cells SGC-7901; cell proliferation was inhibited, invasion and migration of gastric cancer cells were weakened, cell cycle arrest was in S phase, apoptosis of cells obviously increase. CONCLUSION: Nanog gene is closely related to proliferation, cycle, apoptosis, migration and invasion of gastric cancer cells.