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目的探讨p EGFP-N1-NANOGP8真核表达载体及pshRNA-NANOGP8干扰质粒转染人胃癌SGC-7901细胞后,对细胞增殖、周期、凋亡、迁移、侵袭的影响。方法构建p EGFP-NI-NANOGP8真核表达载体及针对人NANOGP8基因的干扰质粒pshRNA-NANOGP8,进行酶切、测序鉴定。将测序正确的质粒在脂质体的介导下转染入SGC-7901细胞,通过荧光显微镜、反转录PCR和Western blot法检测其表达情况,CCK-8法检测SGC-7901细胞增殖情况,流式细胞术分析胃癌细胞周期变化和凋亡情况,TranswellTM实验验证迁移和侵袭能力的变化。结果成功构建出p EGEP-N1-NANOGP8及pshRNA-NANOGP8重组质粒。将重组质粒转染胃癌细胞后,能观察到GFP的表达;反转录PCR结果显示p EGEP-N1-NANOGP8转染组高表达NANOGP8 mRNA,而pshRNA-NANOGP8转染组NANOGP8 mRNA低表达。NANOGP8高表达组能促进肿瘤细胞增殖,促使细胞进入S期,迁移侵袭能力明显增强。而低表达组细胞增殖受抑制,细胞周期停留在G0/G1期,迁移、侵袭能力受抑制。结论逆基因NANOGP8的转录表达,与胃癌的增殖、细胞周期、凋亡、迁移、侵袭有密切的关系。
Objective To investigate the effect of p EGFP-N1-NANOGP8 eukaryotic expression vector and pshRNA-NANOGP8 plasmid on the proliferation, cell cycle, apoptosis, migration and invasion of human gastric cancer SGC-7901 cells. Methods The eukaryotic expression vector p EGFP-NI-NANOGP8 and pshRNA-NANOGP8 targeting human NANOGP8 gene were constructed and identified by restriction enzyme digestion and sequencing. The correct sequencing plasmid was transfected into SGC-7901 cells by lipofectamine. The expression of SGC-7901 cells was detected by fluorescence microscopy, reverse transcription PCR and Western blot. The proliferation of SGC-7901 cells was detected by CCK- Flow cytometry was used to analyze cell cycle changes and apoptosis in gastric cancer cells. TranswellTM assay was used to verify the changes of migration and invasion ability. Results p EGEP-N1-NANOGP8 and pshRNA-NANOGP8 recombinant plasmids were successfully constructed. The expression of GFP was observed after transfection of recombinant plasmids into gastric cancer cells. RT-PCR showed that NANOGP8 mRNA was highly expressed in pEGEP-N1-NANOGP8 transfection group and NANOGP8 mRNA was low in pshRNA-NANOGP8 transfection group. NANOGP8 overexpression group can promote tumor cell proliferation, promote cells into the S phase, migration and invasion was significantly enhanced. The low expression of cell proliferation was inhibited, the cell cycle stays in the G0 / G1 phase, migration, invasion was inhibited. Conclusion The transcriptional expression of the reverse gene NANOGP8 is closely related to the proliferation, cell cycle, apoptosis, migration and invasion of gastric cancer.