VEGF反义寡核苷酸对体外生长的大肠癌HT-29细胞的抑制作用

Inhibitory effect of VEGF antisense oligonucleotide on HT-29 human colorectal cancer cells in vitro

目的:探讨血管内皮生长因子(VEGF)反义寡核苷酸(ASODN)对体外生长的人大肠癌HT-29细胞的抑制作用.方法:实验设空白对照组、脂质体转染组、错义链转染组(SODN组)和不同浓度反义链转染组(ASODN组).用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸(SODN)转染人大肠癌细胞株HT-29,半定量RT-PCR检测各组细胞VEGF mRNA的表达;Western blot测定转染48、72h后VEGF蛋白表达;MTT法和流式细胞术检测细胞增殖和凋亡.结果:转染48h后,ASOND组的VEGF mRNA表达水平明显低于脂质体对照组和SODN组(0.455±0.032vs0.934±0.031,0.915±0.004,P<0.01);脂质体对照组与SODN组之间无显著差异.细胞转染48、72h后,ASODN组蛋白表达明显弱于脂质体对照组和SODN组,且72h弱于48h.与对照组比较,VEGF ASODN对HT-29细胞有明显的生长抑制作用,并且抑制呈剂量和时间依赖性(P<0.05).结论:VEGF ASODN通过抑制VEGF的基因表达,对体外生长的人大肠癌HT-29细胞的增殖进行抑制.

AIM： To investigate the inhibitory effect of vascular endothelial growth factor （VEGF） antisense oligonucleotide （ASODN） on human colorectal cancer cell line HT-29 in vitro. METHODS： Human colorectal cancer HT-29 cells were transfected with VEGF ASODN and scrambled oligodeoxynueleotide （SODN） by LipofectamineTM2000. The expression of VEGF mRNA in HT-29 cells was detected using semiquantitive reverse transcription-polymerase chain reaction （RT-PCR） and the excretion of VEGF protein was measured by Western blot 48and 72 hours after transfection. The proliferation and apoptosis of HT-29 cells were measured using MTT assay and flow cytometry respectively. RESULTS： The expression of VEGF mRNA in HT-29 cells transfected with ASODN was significantly lower 48 h after transfection than that in the plasmid controls or SODN controls （0.455 ± 0.032 vs 0.934 ± 0.031, 0.915 ± 0.004; both P 〈 0.01）. There was no marked difference between the two control groups. The expression of VEGF protein in HT-29 cells was confirmed and the specific band on PVDF membrane in the ASODN group was obviously weaker than that in the control groups. VEGF ASOND inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner （P 〈 0.05）; the apoptotic index of HT-29 cells in the ASODN group was obviously higher than that in the control groups （P 〈 0.05） 72 h after transfection. CONCLUSION： VEGF ASODN can inhibit the proliferation and induce the apoptosis of HT-29 cells in vitro by inhibiting VEGF gene expression.