恩度诱导抑制性VEGFA选择性剪切异构体VEGF165b的表达及其意义

Inhibition effect of Endostar on angiogenesis partly through VEGF165b,the Inhibitory splice variant of VEGFA

目的:探讨重组人血管内皮抑制素(恩度)对人肝癌细胞SMMC-7721VEGF165b表达的影响,及上调VEGF165b表达后,SMMC-7721对恩度敏感性的变化。方法:采用RT-PCR法和蛋白质印迹法检测恩度干预SMMC-7721后VEGF165b的表达变化;稳定转染VEGF165b真核表达质粒到SMMC-7721,RT-PCR和免疫细胞荧光共聚焦显微镜检测VEGF165b、HIF-1α和下游靶基因VEGFA表达的变化;MTT法检测恩度对人肝癌细胞生长抑制率的影响。结果:100和400μg/mL恩度能够诱导VEGF165b mRNA和蛋白表达上调,而外源性VEGF165b下调HIF-1α及下游靶基因VEGFA的表达。转染空质粒后,500和1000μg/mL恩度引起SMMC-7721的生长移植率分别为1.6%和0.4%,转染VEGF165b后,500μg/mL和1000μg/mL恩度引起的生长移植率分别为8.5%(P=0.0000)和20.2%(P=0.0000)。结论:恩度抗肿瘤新生血管的作用部分是通过上调VEGF165b介导的,VEGF165b增加SMMC-7721细胞对恩度的敏感性,两者治疗肿瘤有协同作用。

OBJECTIVE：To investigate the impact of endostar on the expression of VEGF165b,and determine the effect of VEGF165b for he sensitivity of tumor cell to endostar. METHODS：The expression of VEGF165b of SMMC-7721 was detective by RT-PCR and Western Blotting after treated with endostar. After transfected with VEGF165b,the expression of HIF-1α and its downstream target genes VEGFA was analyzed by RT-PCR and Immunofluorescent confocal microscopy. SMMC-7721 cell was transfected with VEGF165b or empty vector,and then treated with endostar,the cell growth inhibiting rate was detected by methly thiazolyl tetrazolium （MTT）. RESULTS：High dose endostar at 100 and 400 μg/mL up-regulated the expression of VEGF165b,while VEGF165b suppressed the expression of HIF-1α and its downstream target genes VEGFA. After transfection with empty-vector,treatment of SMMC-7721 cell with 500 and 1 000 μg/mL endostar induced the tumour cell growth inhibiting rate at 1.6% and 0.4% individually. After transfection with VEGF165b,treatment of SMMC-7721 cell with 500 and 1 000 μg/mL endostar induced the tumour cell growth inhibiting rate at 8.5% （P=0.000 0） and 20.2%（ P=0.000 0） individually. CONCLUSIONS：Endostar can inhibite tumour growth and anti-angiogenisis partly via VEGF165b,and VEGF165b sensitize the tumour cell to endostar. VEGF165b and endostar have synergistic effect on tumor suppression.