反义寡核苷酸抑制神经母细胞瘤LA-N-5细胞血管内皮生长因子mRNA表达及其生物效应的研究

摘要 目的 研究血管内皮生长因子（VEGF）反义寡核苷酸（ASODN）转染对神经母细胞瘤LA-N-5细胞VEGF mRNA表达的影响以及对肿瘤细胞增殖、分化的影响。方法 用LipofectamineTM2000介导的VEGF ASODN和错义寡核苷酸（MSODN）转染LA-N-5细胞，半定量RT-PCR检测各组细胞VEGF165和VEGF121 mRNA转染前后不同时间表达的变化；MTT法测定转染后各组细胞的生长曲线及抑制率。结果 半定量RT-PCR检测结果显示，在转染后72 h VEGF165和VEGF121 mRNA的表达：ASODN组为0.346±0.029和0.227±0.036，ASODN+LipofectamineTM2000组为0.275±0.035和0.165±0.017。ASODN组和ASODN+LipofectamineTM2000组均显著抑制VEGF mRNA的表达，ASODN+LipofectamineTM2000组抑制作用较ASODN组更强（P<0.05）；转染后ASODN组和ASODN+LipofectamineTM2000组细胞增殖显著受抑，在48 h时抑制率最高，分别为（39.92±2.74）%及（55.80±2.52）%,ASODN+LipofectamineTM2000组对细胞增殖的抑制作用较ASODN组更为明显（P<0.05）。MSODN组、MSODN+LipofectamineTM2000组对LA-N-5细胞增殖抑制作用与对照组差异无统计学意义（P>0.05）。结论 VEGF ASODN可抑制神经母细胞瘤LA-N-5细胞VEGF在mRNA水平的表达，并能抑制神经母细胞瘤LA N 5细胞的增殖和分化，LipofectamineTM2000能明显增强VEGF ASODN的抑制效果。

A study of inhibition on expression of VEGF by antisense oligonucleotides and its biological effect on LA-N-5 cells

Abstract ObjectiveT o investigate if VEGF ASODN transfection to LA-N-5 cells inhibits VEGF mRNA expression, thus affects its differentiation and proliferation.Methods LA-N-5 cells were transfected with VEGF ASODN or MSODN alone, ordelivered by LipofectamineTM2000, respectively, VEGF mRNA pre- and post transfection expression was detected by semi-quantitative RT-PCR, MTT was employed for evaluation of differentiation and proliferation of cells in each group.Results ①The semi quantitative RT-PCR detection showed VEGF165 and VEGF121 mRNA expression were of 0.275±0.035, and 0.165±0.017 respectively in group of ASODN+LipofectamineTM2000, compared to 0.346±0.029 and 0.227±0.036 respectively in group of ASODN, after 72 hours.VEGF mRNA expression in groups of ASODN and ASODN+ LipofectamineTM2000 were significantly inhibited and the inhibition with ASODN+LipofectamineTM2000 was more significant compared with that in ASODN (P<0.05）.②MTT assay at 0,12, 24, 36, 48, 60, and 72 hours after transfection showed that cellular proliferation was significantly inhibited in the groups of ASODN and ASODN+LipofectamineTM2000.After 48 hours the inhibition rate reached （39.92±2.74）% and（55.80±2.52）%, especially in later group, the inhibition was more significant than that in ASODN group（t=-12.065,P=0.00, P<0.05）. The growth and proliferation of LA-N-5 cells in groups of MSODN and MSODN+ LipofectamineTM2000 did not show any difference compared to control group（P>0.05）.Conclusions VEGF ASODN is capable of suppressing VEGF mRNA expression and cellular proliferation in LA-N-5 cells, and LipofectamineTM2000 can significantly strengthen this inhibition.