重组人内皮抑素与血管内皮生长抑制因子融合基因对角膜新生血管内皮细胞的协同作用

Effect of recombinant adenovirus-mediated endostatin-soluble vascular endothelium growth inhibitor fusion gene on corneal neovascularization

目的:观察内源性血管抑制因子内皮抑素与血管内皮生长抑制因子融合基因对新生血管内皮细胞增殖的协同抑制作用,分析其作用特异性。方法:实验于2002-06/2003-06在解放军第二军医大学微生物实验室完成。常规分子生物学构建携带融合基因内皮抑素-血管内皮生长因子的重组腺病毒,酶切法、聚合酶链反应法鉴定重组腺病毒-内皮抑素-血管内皮生长因子的正确性,半数细胞感染剂量法测定重组腺病毒滴度。以磷酸盐缓冲液、重组腺病毒-β-半乳糖苷酶和重组腺病毒-血管内皮生长因子151为对照组,病毒滴度均为感染复数(MOI)=20,体外感染人静脉内皮细胞ECV304与小鼠成纤维细胞L9294h,72h后应用反转录-聚合酶链反应和Western印迹法分别检测融合基因细胞内的转录及表达情况;于病毒感染后第1,2,3,4,5和7天,结晶紫染色法检测活细胞数,用570/630nm吸光度(A)值表示,绘制并比较ECV304和L929转基因细胞的生长曲线,观察其作用并分析其作用特异性。结果:①聚合酶链反应法证实成功构建了融合基因重组腺病毒-内皮抑素-血管内皮生长因子,半数细胞感染剂量法测得病毒滴度为2×108pfu/mL。②各组ECV304细胞结晶紫染色检测结果比较:除感染第1天重组腺病毒-血管内皮生长因子151感染组外(P>0.05),其余感染各时相重组腺病毒-血管内皮生长因子151感染组、重组腺病毒-β-半乳糖苷酶感染组及磷酸盐缓冲液组ECV304细胞A值均显著高于重组腺病毒-内皮抑素-血管内皮生长因子感染组(P<0.01)。③各组L929细胞结晶紫染色检测结果比较:重组腺病毒-内皮抑素-血管内皮生长因子感染组、重组腺病毒-β-半乳糖苷酶感染组及磷酸盐缓冲液组L929细胞的A值两两比较,差异均无显著性意义(P>0.05)。结论:重组腺病毒-内皮抑素-血管内皮生长因子在体外能有效表达具有生物学活性的融合基因产物,对静脉内皮细胞的增殖具特异性抑制作用,且较单基因产物的抑制作用更强,可用于治疗角膜新生血管的进一步体内实验。

AIM： To observe the inhibitory and special effects of recombinant adenovirus-mediated endostatin-soluble vascular endothelium growth inhibitor （sVEGI） fusion gene （Ad-hENDO-sVEGI） on proliferation of vascular endothelium cell line. METHODS： The experiment was carried out in the laboratory of the Department of Microbiology of the Second Military Medical University of Chinese PLA between June 2002 and June 2003. Recombinant adenovirusmediated endastatin-soluble vascular endothelium growth inhibitor fusion gene was constructed by routine molecular biology, and identified by enzyme digestion and polymerase chain reaction （PCR）. The viral particles were titered by 50% tissue culture infective dose method. Phosphate buffered solution （PBS）, Ad-LacZ and Ad-VEGI151 were regarded as control groups with virus titer of MOI=20. When ECV304 and L929 were infected for 4 hours in vitro, the expression of fusion gene mRNA and protein were demonstrated by RT-PCR and Western blot respectively. The bioactivity was determined by Violet crystal assay on day 1, 2, 3, 4, 5 and 7 and represented by absorbance （A） 570/630 nm; the growth curve of ECV304 and L929 was drawn and compared to observe its effects and the specificity. RESULTS： ①PCR showed that the fusion gene was successfully cloned and tittered 2×^8 pfu/mL. ②Comparison of violet crystal assay results of ECV304 cells in each group： Except the 1st day of infection of the AdVEGI151 group （P 〉 0.05）, the A values of ECV304 cells of the Ad-VEGI 151, PBS and Ad-LacZ groups were all higher than the Ad-hENDO-sVEGI group （P 〈 0.01）. ③Comparison of violet crystal assay results of L929 cell of each group： No significant difference was found in the A values of L929 cells among the Ad-hENDO-sVEGI group, PBS group and Ad-LacZ group during group comparison （P 〉 0.05）. CONCLUSION： The constructed Ad-hENDO-sVEGI can express fuse gene products with bioactivity, and shows a specific synergistic inhibition on proliferation of endothelial cell of vein, which is stronger than that of monogenic products. It can be used in further experiments for treating corneal neovascularization.