论文部分内容阅读
目的验证分化抑制因子1(Id-1)在环氧合酶2(COX-2)介导的胃癌血管生成中的作用及机制。方法建立高表达 COX-2和表达 COX-2特异性 siRNA 的胃癌 SGC7901细胞,通过Western 印迹和逆转录 PCR 方法检测两种亚细胞系中 Id1的表达,ELISA 方法分析两种细胞上清中血管内皮生长因子(VEGF)的表达差异。通过四唑盐比色(MTY)实验分析两种细胞亚系的上清对体外脐静脉内皮细胞(HU-LT)增殖的影响,并在 SGC7901/COX-2中抑制 Id1后观察培养上清中 VEGF 的变化以及对脐静脉内皮细胞(HU-LT)增殖的影响。裸鼠成瘤实验观察转染细胞的成瘤性及肿瘤新生微血管密度(MVD)。结果成功建立高表达 COX-2以及表达特异性 COX-2小干扰 RNA(siRNA)的稳定克隆,并鉴定了不同克隆的转染效率。高表达 COX-2的胃癌 SGC7901细胞中 Id1的蛋白及mRNA 表达水平增高,而转染 COX-2-siRNA 的 SGC7901细胞中 Id1的表达则均低于对照组。COX-2高表达的 SGC7901/COX-2细胞上清中 VEGF 的蛋白含量高于对照组(2060±42 vs 1248±28,P=0.000),而 SGC7901/COX-2-siRNA 细胞上清中 VEGF 的蛋白含量低于对照组(1024±20,2033±27 vsP=0.000)。在 SGC7901/COX-2细胞条件培养基中,HU-LT 细胞生长较对照组快;在 SGC7901/COX-2-SiRNA 细胞条件培养基中,HU-LT 细胞生长较对照组缓。在 SGC7901/COX-2中抑制 Id1后,培养上清中 VEGF 的含量增加以及对 HU-LT 促进增殖的作用均被逆转。裸鼠成瘤试验显示抑制 Id1后SGC7901/COX-2细胞成瘤性降低[(353±12)mg vs(1020±91)mg,P=0.038],MVD 降低(8.8±1.6vs 20±1.7,P=0.001)。结论 COX-2可以上调 SGC7901细胞中 Id1的表达,并通过此途径影响内皮细胞的体外增殖能力。因此在胃癌发生发展中 Id1参与了 COX-2所介导的促血管生成过程,有可能成为胃癌抗血管治疗的新靶标。
Objective To investigate the role and mechanism of Id-1 in angiogenesis induced by COX-2 in gastric cancer. Methods Gastric cancer SGC7901 cells with high expression of COX-2 and COX-2 specific siRNA were established. The expression of Id1 in both sub-cell lines was detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) Differences in the expression of growth factor (VEGF). The proliferation of human umbilical vein endothelial cells (HU-LT) in vitro was examined by MTT assay and the inhibition of Id1 in SGC7901 / COX-2 cells was observed in culture supernatant VEGF changes and the effects on the proliferation of umbilical vein endothelial cells (HU-LT). The tumorigenicity of the nude mice was observed to observe the tumorigenicity and the neovascularization density (MVD) of the transfected cells. RESULTS: Stable clones highly expressing COX-2 and expressing specific COX-2 small interfering RNA (siRNA) were successfully established and the transfection efficiency of different clones was identified. Id1 protein and mRNA expression increased in gastric cancer SGC7901 cells overexpressing COX-2, while the expression of Id1 in SGC7901 cells transfected with COX-2-siRNA was lower than that in control group. The protein content of VEGF in the supernatant of COX-2-overexpressing SGC7901 / COX-2 cells was higher than that in the control group (2060 ± 42 vs 1248 ± 28, P = 0.000), while the expression of VEGF in SGC7901 / COX- Protein content was lower than the control group (1024 ± 20,2033 ± 27 vsP = 0.000). In SGC7901 / COX-2 cell conditioned medium, HU-LT cells grew faster than the control group; in SGC7901 / COX-2-SiRNA cell conditioned medium, HU-LT cells grew slower than the control group. After Id1 was inhibited in SGC7901 / COX-2, the increase of VEGF content in culture supernatant and the effect of promoting the proliferation of HU-LT were reversed. The tumorigenicity of SGC7901 / COX-2 cells was inhibited by Id1 ([353 ± 12] mg vs (1020 ± 91) mg, P = 0.038] and MVD decreased by 8.8 ± 1.6 vs 20 ± 1.7 P = 0.001). Conclusion COX-2 can up-regulate the expression of Id1 in SGC7901 cells and affect the proliferation of endothelial cells in vitro through this pathway. Therefore, Id1 is involved in the process of angiogenesis mediated by COX-2 in the development of gastric cancer, which may become a new target of anti-angiogenesis in gastric cancer.