内皮抑素-血管内皮细胞抑制因子重组腺病毒对新生血管的抑制效应

Inhibitory effects of endostatin-vascular endothelial growth inhibitor recombinant adenoviruses on neovascularization

内皮抑素与血管内皮细胞抑制因子均为内源性新生血管抑制分子。为了探讨两分子融合后的生物学功能,以重组腺病毒为载体介导融合基因在体内外表达。体外实验表明重组腺病毒Ad-hENDO-VEGI151能够在ECV304、HepG2、L929等细胞中表达41000大小的融合蛋白,对血管内皮细胞增殖有明显的特异性抑制作用,对HepG2和L929细胞无抑制作用(F=13112.13,P=0.0001)。体内实验表明Ad-hENDO-VEGI151重组腺病毒表达的融合蛋白可以显著抑制鸡胚绒毛尿囊膜血管的生成;与AdLacZ对照组相比,Ad-hENDO-VEGI151对兔炎症性角膜新生血管的抑制明显(F=1413.11P=0.0001),免疫组化结果显示,融合蛋白主要在角膜上皮层表达;治疗荷肝癌裸鼠,注射Ad-hENDO-VEGI151的肿瘤体积(487.7±241.2mm3)与AdLacZ对照组(4075.9±1849.9mm3)差异显著(F=14.80P=0.0085),抑瘤率达到88.03%,免疫组化结果显示,胞膜染色阳性,Ad-hENDO-VEGI151组肿瘤的微血管密度30.75±3.31%与AdLacZ组50.25±8.65%差异明显F=17.72P=0.0056抑制率为39%。结果提示:重组腺病毒Ad-hENDO-VEGI151能够表达有生物学活性的融合蛋白,并发挥特异性的新生血管抑制作用。

Endostatin and vascular endothelial growth inhibitor are endogenous angiostatic molecules.We use the re-combinant adenoviruses expressing the endostatin-VEGI 151 fusion protein to treatment three cell lines and three ani-mal models to investigate the biological activity of the fusion molecular.Western blot detected the fusion protein about 41kD could be expressed when Ad-hENDO-VEGI 151 affected the cell ECV304,HepG2and L929,respectively.The expressed fusion protein showed a specific inhibition on the proliferation of ECV304cell,but showed no in-hibitory effect on growth of HepG2and L929cell(F=13112.13,P=0.0001).On chick choriallantic membrane(CAM)as-say,the expressed fusion protein markedly inhibited the neovascularization.Inflammatory corneal neovascularization(CNV)in rabbit induced by placement of intrastromal sutures results in a uniform neovascular response.On this model,direct subconjunctival injection of Ad-hENDO-VEGI 151 could express the fusion protein in vivo and suppress the development of CNV.Analyzing the CNV area found that topical application of Ad-hENDO-VEGI 151 led to a significant suppression of CNV(F=1413.11,P=0.0001),compared with control groups AdLacZ.Immunohistochemical staining showed the fusion protein expressed mainly in corneal epithelium.Compared with the control group AdLacZ(4075.9±1849.9mm 3 ),the average tumor size of group AdCA13-hENDO-VEGI 151 was reduced in size(487.7±241.2mm 3 )(F=14.80,P=0.0085)with an inhibition of rate88.03%.Immunohistochemical staining showed the aden-oviruses carried the fusion gene could be expressed on liver cancer cell membrane.The MVD was decreased in the treated mice(30.75±3.31%)than in the AdLacZ control(50.25±8.65%)(F=17.72,P=0.0056)with an inhibition of rate39%.These results suggest that the fuion protein expressed by the recombinant adenovirus have significant effect on inhibiting the neovascularization specifically.