阻断Notch信号途径能有效抑制A549细胞增殖

摘要

目的 Delta-like 4(DLL4)是Notch信号通路的配体之一,是近年新发现的血管生长调控因子,研究表明DLL4的高表达与肺腺癌转移预后密切相关.该文旨在研究DLL4在A549细胞中的表达,以及DLL4基因表达对人A549细胞增殖与凋亡的影响.方法 (1)体外培养A549细胞,免疫细胞化学法检测DLL4在A549细胞中的表达及定位;(2)设计并化学合成靶向DLL4基因的siRNA序列,lipofectamineTM2000介导DLL4 siRNA转染A549细胞,RT-PCR进行DLL4-mRNA定量,Western blot分别检测A549细胞DLL4蛋白的改变;(3)四甲基偶氮唑盐(MTT)比色法检测细胞的存活和生长变化;(4)激光共聚焦显微镜检测Annexin V-FITC标记的A549细胞的细胞凋亡比率.结果 (1)A549细胞表达DLL4蛋白,且定位于胞浆中;(2)DLL4-mRNA和DLL4蛋白的表达在对照组与干扰组之间有显著差异(P<0.05);(3)MTT检测显示抑制DLL4表达对A549细胞的增殖有抑制作用,DLL4干扰组细胞增殖抑制率为43.85%,未处理组细胞增殖抑制率为23.81%;(4)抑制DLL4基因表达,A549细胞的凋亡比率明显升高,DLL4干扰组细胞凋亡率为(25.01±4.32)%,未处理组细胞凋亡率为(14.24±0.98)%.结论 DLL4在A549细胞表达.特异性阻断DLL4/Notch信号途径能有效抑制A549细胞增殖,促进细胞凋亡.%Objective To detect the expression of DLL4 in human lung adenocarcinoma A549 cells and discovery the impact of DLL4 on proliferation and apoptosis in A549 cells. Methods Delta-like 4 expression was detected by immunohistochemical staining in A549 cells ;The siRNAs targeting to DLL4 gene were designed and chemically synthesized. DLL4 siRNAs were transfected into A549 cells by lepofectamine·2000. Total RNA was extracted from A549 cells. RT-PCR agar gel electrophoresis was performed to detect the expression of DLL4 mRNA. Total cellular protein of treated cells was extracted and analyzed by polyacrylamide gel electrophoresis. The treatments were divided into two groups which were DLL4 siRNA-duplex group and no treatment group. The impact of DLL4 siRNA on A549 cells proliferation was detected by MTT assay. The treatments were divided in to 4 groups including DLL4 siRNA-duplex group, DLL4 siRNA-scrambled negative control group, lepofectamine group and no treatment group. The proportion of apoptotic cells was counted by laser scanning confocal microscope with Annexin V-FITC staining marked. The treatments were divided into DLL4 siRNA-duplex group and no treatment group. Results The expression of DLL4 protein was detected in cytoplasm of A549 cells. The results of RT-PCR and Western blot indicated that DLL4 was expressed in A549 cells at levels of mRNA and protein,which had significant difference between DLL4 siRNA-duplex group and no treatment group(P <0. 05) . MTT results showed that the proliferation ratio was 43. 85% in the DLL4 siRNA-duplex group,which was 23.81% in the no treatment group. DLL4 siRNAs accelerated apoptosis of A549 cells. The apoptosis ratio was (25.01 ±4.32)% in the DLL4 siRNA-duplex group,which was (14. 24 ± 0. 98) % in the no treatment group. Conclusion DLL4 can be expressed in A549 cells. DLL4 siRNAs inhibit the expression of DLL4 in A549 cells, meanwhile inhibit A549 cells proliferation, and accelerate A549 cells apoptosis.