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目的:构建T F1基因干扰重组质粒及建立T F1基因干扰瞬时转染胃癌细胞模型,然后通过干扰目标胃癌细胞中的T F1基因来检测T F1对胃癌细胞的生物学功能的影响。方法:首先通过real-time PCR和Western印迹验证T F1在胃癌细胞系BGC823,SGC7901,MGC803中的表达,筛选出T F1表达量最高的一株细胞作为干扰转染的目标细胞;设计及构建3个T F1的shRNA表达载体pLVX-shRNA-T F1-shRNA1~3,将其分别转染至目标胃癌细胞系中,应用real-time PCR和Western印迹筛选出干扰效率最强的shRNA,最后通过M及流式细胞术检测T F1对胃癌细胞凋亡能力的影响,通过Transwell小室迁移实验来检测对其迁移功能的影响。结果:与GES-1比较,T F1基因在BGC823,SGC7901,MGC803中的表达均有上调(P<0.05),其中以BGC823表达最高;3个T F1shRNA对T F1基因均有抑制作用,以pLVX-shRNA-T F1-shRNA2干扰效果最强;干扰胃癌细胞BGC823中的T F1可使细胞生长活力减弱,凋亡明显增加,但对其迁移功能无明显影响。结论:T F1基因在胃癌细胞系BGC823中上调最明显;pLVX-shRNA-T F1-shRNA2可成功干扰BGC823中T F1的表达;T F1可抑制BGC823细胞的凋亡。
OBJECTIVE: To construct T F1 gene interference recombinant plasmid and establish T F1 gene to interfere with transient transfection of gastric cancer cell model, and then detect the effect of T F1 on the biological function of gastric cancer cells by interfering with T F1 gene in target gastric cancer cells. METHODS: The expression of T F1 in gastric cancer cell lines BGC823, SGC7901 and MGC803 was verified by real-time PCR and Western blotting. One strain with the highest expression level of T F1 was screened out as the target cell for transfection. The design and construction of 3 T F1 shRNA expression vector pLVX-shRNA-T F1-shRNA1 ~ 3, were transfected into the target gastric cancer cell lines, the use of real-time PCR and Western blot screening out the strongest interference shRNA, and finally by M Flow cytometry was used to detect the effect of T F1 on the apoptosis of gastric cancer cells. The effect of T F1 on the migration of gastric cancer cells was tested by Transwell chamber migration assay. Results: Compared with GES-1, the expression of T F1 gene in BGC823, SGC7901 and MGC803 was up-regulated (P <0.05), and the expression of T F1 gene was the highest in BGC823. -shRNA-T F1-shRNA2. The interference of T F1 in gastric cancer cell BGC823 attenuated the cell viability and increased the apoptosis, but had no significant effect on its migration function. CONCLUSION: T F1 gene is most significantly up-regulated in gastric cancer cell line BGC823. PLVX-shRNA-T F1-shRNA2 can successfully interfere with the expression of T F1 in BGC823. T F1 can inhibit the apoptosis of BGC823 cells.