论文部分内容阅读
目的 将构建的Fn14短发夹RNA (short hairpin RNA,shRNA)转染胰腺癌PANC-1细胞并检测接种转染的癌细胞增殖情况,分析探讨肿瘤坏死因子相关弱诱导细胞凋亡/成纤维细胞生长因子诱导因子14 (TNF-related weak inducer of apoptosis/fibroblast growth Factor-inducible 14,TWEAK/Fn14)在胰腺癌细胞增殖过程中的作用.方法 构建靶向Fn14基因的shRNA并转染胰腺癌PANC-1细胞来特异性沉默Fn14基因的表达.采用流式细胞术及免疫荧光法检测shRNA干扰序列对Fnl4表达的影响;采用CCK-8法检测阻断TWEAK/Fn14信号通路后PANC-1肿瘤细胞增殖情况;采用Western blotting法检测阻断TWEAK/Fn14信号通路后下游因子核因子κB (nuclear factor-κB,NF-κB)、TWEAK及半胱氨酸天冬氨酸蛋白酶-3(caspase-3)蛋白表达的影响来进一步探索该信号通路的作用机制.结果 转染Fn14 shRNA 24 h后,PANC-1肿瘤细胞的吸光度值(A值)显著低于对照组(P＜0.000 1);质粒转染后PANC-1细胞的NF-κB、TWEAK及caspase-3蛋白表达量也明显低于对照组(P＜0.05);且抑制TWEAK/Fn14信号通路后PANC-1细胞的凋亡增加.结论 TWEAK/Fn14参与了胰腺癌的发生发展过程.TWEAK/Fn14可影响胰腺癌肿瘤细胞的凋亡.“,”Objective To investigate the effect of TNF-related weak inducer of apoptosis/fibroblast growth Factor-inducible 14 (TWEAK/Fn14) on the cell proliferation by transfecting Fn14 shRNA to PANC-1 cells.Methods The shRNA gene targeting Fn14 gene was constructed and transfected into pancreatic cancer cell line PANC-1 to specifically silence the expression of Fn14 gene.The effect of shRNA interference sequence on the expression of Fn14 was detected by flow cytometry and immunofluorescence.CCK-8 was used to detect the cell proliferation of PANC-1 after blocking TWEAK-induced signal pathway.Western blotting method was used to detect the expressions of downstream factors such as nuclear factor-kappa B (NF-κB),TWEAK and caspase-3 to explore the pathway mechanism of TWEAK/Fn14.Results The absorbance value (A value) in the Fn14 shRNA group was significantly lower than the control groups in 24 hours after transfected (P＜0.000 1).After the specific shRNA sequences transfected PANC-1 cells,NF-κB,TWEAK and caspase-3 protein expressions were also significantly lower than the control group (P＜0.05),and the apoptosis of PANC-1 cells increased after inhibition of TWEAK/Fn14 signaling pathway.Conclusions TWEAK/Fn14 involved in the progression of pancreatic cancer.The Fnl4 expression could influence the process of cell apoptosis.