目的研究胃泌素对人脐静脉血管内皮细胞(HUVEC)体外增殖和管状结构形成的作用。方法免疫细胞化学等检测HUVEC中胃泌素受体(CCK—BR)的表达;用10和100nmoL胃泌素处理HUVEC后,MTT和软琼脂克隆形成实验检测细胞增殖能力,小管形成实验观察HUVEC管状结构生成情况;实时荧光定量PCR和酶联免疫吸附法检测HUVEC中血管内皮生长因子(VEGF)和低氧诱导因子.1a(HIF,la)基因的表达。结果HUVEC表达CCK—BR,经10和100nmoL胃泌素处理后.细胞增殖率和克隆形成率显著增加(P〈0.01),细胞中VEGF和HIF-1a基因的表达量高于对照组(P〈0.01),HUVEC形成的半环状和环状管状结构数也高于对照组(P〈0.01)。结论胃泌素与HUVEC表达的CCK.BR结合后,上调VEGF和HIF.1a基因的表达,促进HUVEC体外增殖及管状结构的形成能力。
Objective To study the effect of gastrin on the proliferation and angiogenesis of human umbilical vascular endothelial (HUVE) cell in vitro. Methods The immunocytochemistry assay, realtime-PCR, and Western blot were used to detect the gastrin receptor (CCK-BR) expression in HUVE cells. After HUVE cells were treated with 10 and 100 nmol/L gastrin for 72 h, MTT and soft agar colony formation assay were used to test the cell proliferation rate and colony formation rate, and the vascular-like structures were observed by three-dimensional culture of HUVE cells. The half-ring and ring vascular numbers were counted with five random visions. The vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-la (HIF-la) mRNA and protein levels in HUVE cells were detected by realtime PCR and ELISA assay respectively. Results HUVE cells expressed CCK-BR. After the treatment with 10 and 100 nmol/L gastrin, the cell proliferation rate was increased by 48.48% and 82.82 % compared to the control group, and colony formation rate was increased to 31.33% and 45.67 % as compared with 15.33% in the control group( P〈0. O1 ). Relative expression quantities of VEGF and HIF-la genes were 2.3 and 4.6 folds (VEGF) and 20.76 and 26.77 folds ( HIF-1 a) than those in the control group. The concentration of VEGF protein in culture medium was 22l and 392 Ixg/mg protein higher than that in control group. The numbers of half-ring and ring vascular structures were ( 14. 00 ± 3.130, 39.33 ±7.57 and 34.33 ±4.50 )/vision and ( 8.33 ± 2.51,41.33 ±5.85 and 37.67 ±3.51 )/vision in control, 10 and 100 nmol/L gastrin-treated groups, respectively ( P〈O. O1 ). Conclusion Gastrin up-regulates the expression of VEGF and HIF-1 a genes in HUVE cells and promotes cell proliferation and vascular-like structure formation of HUVE cells in vitro by being combined to CCK-BR, which may be involved in the development and metastasis of gastric cancer.