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目的:探讨14-3-3σ过表达对胰腺癌PANC-1细胞侵袭能力的影响。方法:以人基因组为模板,通过PCR扩增出14-3-3σ基因的编码序列,定向插入真核表达载体pEGFP-N1中,构建重组质粒pEGFP-14-3-3σ。经酶切和测序验证后,首先将pEGFP-14-3-3σ质粒转染HEK293T细胞观察转染效率;然后用脂质体法稳定转染PANC-1细胞,并以转染空载体及未转染的PANC-1细胞分别作为阴性对照和空白对照,用实时荧光定量PCR和Western blot法分别检测目的基因mRNA和蛋白表达情况,Transwell法检测细胞侵袭能力。结果:酶切和序列测定证实14-3-3σ基因正确插入pEGFP-N1载体中,pEGFP-14-3-3σ对HEK293T细胞的转染效率达65%。PANC-1细胞转染pEGFP-14-3-3σ后,14-3-3σmRNA与蛋白表达水平均明显增高;Transwell侵袭实验结果显示,转染pEGFP-14-3-3σ的PANC-1细胞穿膜数较转转染空载体及未转染的PANC-1细胞明显增多(129.4±19.6 vs.76.4±17.7,78.7±16.7)(均P<0.05)。结论:14-3-3σ基因过表达能增强胰腺癌PANC-1细胞的侵袭能力。
Objective: To investigate the effect of 14-3-3σ overexpression on the invasiveness of pancreatic cancer PANC-1 cells. Methods: The coding sequence of 14-3-3σ gene was amplified by PCR using the human genome as a template and inserted into the eukaryotic expression vector pEGFP-N1 to construct the recombinant plasmid pEGFP-14-3-3σ. After digestion and sequencing, pEGFP-14-3-3σ plasmid was transfected into HEK293T cells to observe the transfection efficiency. Then, PANC-1 cells were stably transfected by liposome and transfected into empty vector and untransfected The transfected PANC-1 cells were used as negative control and blank control, respectively. The mRNA and protein expression of target gene were detected by real-time fluorescence quantitative PCR and Western blot respectively. Transwell assay was used to detect cell invasion. Results: The restriction endonuclease digestion and sequence analysis confirmed that the 14-3-3σ gene was correctly inserted into pEGFP-N1 vector. The transfection efficiency of pEGFP-14-3-3σ to HEK293T cells was 65%. PANC-1 cells transfected with pEGFP-14-3-3σ, 14-3-3σmRNA and protein expression levels were significantly increased; Transwell invasion assay showed that transfected pEGFP-14-3-3σ PANC-1 cells through the membrane (129.4 ± 19.6 vs.76.4 ± 17.7, 78.7 ± 16.7) (P <0.05) compared with those in transfected empty vector and untransfected PANC-1 cells. Conclusion: Overexpression of 14-3-3σ enhances the invasiveness of pancreatic cancer PANC-1 cells.