VEGF反义寡核苷酸对人胆囊癌细胞VEGF、Flt-1及KDR mRNA和蛋白水平表达的影响

目的 体外研究Oligofectamine介导的血管内皮生长因子（VEGF）反义寡核苷酸（ASODN）转染对人胆囊癌细胞VEGF、Fit-1及KDR在mRNA和蛋白水平的表达及生长增殖和凋亡的影响。方法运用Oligofectamine介导的VEGFASODN和SODN转染人胆囊癌细胞GBC-SD,半定量RT-PCR检测各组细胞VEGF、Fit-1及KDRmRNA转染前后不同时间的表达变化、ELISA测定转染后各组细胞培养上清液VEGF蛋白浓度。结果ASODN组及ASODN＋Oligofectamine组都能显著抑制VEGF、Fit-1及KDRmRNA的表达,且VEGFASODN＋Oligofectamine的抑制作用比ASODN更强（P〉0．05）。ELISA测定结果显示ASODN组及ASODN＋Oligofectamine组均抑制VEGF蛋白的分泌（P〈0．05）,且ASODN＋Oligofectamine组的VEGF蛋白浓度低于ASODN组（P〉0．05）。结论VEGFASODN能抑制人胆囊癌细胞VEGF、Fit-1和KDR在mRNA水平的表达和VEGF蛋白的表达,从而促进GBC-SD细胞的凋亡;Oligofectamine能明显增强VEGFASODN的抑制效果。

Effects of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide on mRNA and expression of VEGF, flt-1, and kinase insert domain containing receptor and VEGF excretion in human gallbladder carcinoma cells

OBJECTIVE: To investigate the effects of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ASODN) on the mRNA and protein expression of VEGF, Flt-1, and kinase insert domain containing receptor (KDR) and VEGF excretion in human gallbladder carcinoma cells. METHODS: Human gallbladder carcinoma cells of the line GBC-SD were cultured and transfected with VEGF ASODN and sense oligodeoxynucleotide (SODN) mediated by Oligofectamine. The toxicity of SODN and Oligofectamine to the GBC-SD cells was examined by MTT method. RT-PCR was used to detect the mRNA and expression of VEGF, Flt-1, and KDR, and ELISA was used to detect the protein expression of VEGF. RESULTS: MTT method showed that SODN and Oligofectamine were not toxic to the GBC-SD cells. The mRNA expression levels of VEGF, Flt-1, and KDR of the ASODN and ASODN + Oligofectamine groups were all significantly lower than those of the control group (all P < 0.05), and were the lowest 72 hours after transfection, and then gradually increased. ELISA showed that there were not significant differences in the VEGF protein concentration in the supernatant of the GBC-SD cells among the SODN, SODN + Oligofectamine, and control groups (all P < 0.05), however, the VEGF protein concentration in the supernatant of the GBC-SD cells of the ASDN and ASDN + Oligofectamine groups were significantly lower than that of the control group (both P < 0.05). CONCLUSION: VEGF ASODN inhibits the mRNA and protein expression of VEGF, Flt-1, and KDR and VEGF excretion in human gallbladder carcinoma cells. Oligofectamine strengthens the effect of ASODN.