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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(TNFrelated 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Fn14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 construct the short hairpin RNA (shRNA) of Fn14 transfected into pancreatic cancer PANC-1 cells and detect the proliferation of the transfected cancer cells, and to explore whether tumor necrosis factor-related weak induction of apoptosis / fibroblasts The role of TNFrelated weak inducer of apoptosis / fibroblast growth factor-inducible 14 (TWEAK / Fn14) in the proliferation of pancreatic cancer cells. Methods shRNA targeting Fn14 gene was constructed and transfected into pancreatic cancer PANC-1 cells to specifically silence Fn14 gene expression. The effect of shRNA interference sequence on Fn14 expression was detected by flow cytometry and immunofluorescence. The proliferation of PANC-1 tumor cells was detected by CCK-8 assay. The expression of TWEAK / Fn14 signaling pathway downstream factors NF-κB (NF-κB), TWEAK and caspase-3 protein expression to further explore the role of the signal pathway mechanism. Results After transfected with Fn14 shRNA for 24 h, the absorbance value (A) of PANC-1 tumor cells was significantly lower than that of the control group (P <0.000 1). The NF-κB, TWEAK and caspase- 3 protein expression was also significantly lower than the control group (P <0.05); and inhibition of TWEAK / Fn14 signaling pathway PANC-1 cells increased apoptosis. Conclusion TWEAK / Fn14 is involved in the development of pancreatic cancer. TWEAK / Fn14 can affect the apoptosis of pancreatic cancer cells.