目的观察人血管内皮生长因子165(hVEGF_(165))与嵌合水蛭肽融合基因(fusedhirudin,FH)对兔颈动脉损伤后再狭窄的防治作用,为再狭窄的基因治疗提供新的思路。方法通过基因重组构建 hVEGF_(165)与 FH 的融合基因(hVEGF_(165)-FH)。制备兔颈总动脉血管成形术损伤后再狭窄模型,损伤局部转染该融合基因作为治疗组。同时局部转染 pcDNA3.0空质粒作为对照组,通过血管造影、病理分析等观察融合基因是否抑制兔颈总动脉血管成形术后再狭窄。结果局部转染融合基因后1周和3周,造影显示治疗组血管狭窄程度与对照组比较显著减轻;病理分析显示。治疗组较对照组管腔狭窄程度显著减轻(1周:11.50% vs 33.25%;3周:19.75% vs 52.25%,P<0.05);治疗组较对照组内膜增生程度(内膜/中膜,I/M)显著减轻(1周:0.12 vs 0.50;3周:0.35 vs 1.07,P<0.05)。同时融合基因对血管损伤处的血栓形成有抑制作用,但对系统凝血指标无影响,无全身出血并发症。结论融合基因 hVEGF_(165)-FH 可有效地预防兔颈动脉成形术后再狭窄。以内皮修复为基础,构建双靶点甚至多靶点的融合基因可能是未来再狭窄防治的一个有效策略。
A fused hirudin peptide was designed, synthesized and purified and its antithrombin and antiplatelet functions were tested.This bifunctional anticoagulation peptide was composed of three inhibitory regions: (1)the tail of hirudin (hirudin 53-64 ), a potent direct antithrombin , binds to the anion binding exosite (ABE) of thrombin.Hirudin 53-64 blocks fibrinogen recognition site of thrombin and consequently inhibits thrombin directly;(2) Arg -Pro -Pro -Gly - Phe (RPPGF) amino acid sequence, a metabolite of bradykinin,binds to the active site of thrombin and thereby inhibits the amidolytic activity of thrombin;(3)Arg-Gly-Asp(RGD)amino acid sequence, an inhibitor of GP IIb/IIIa, blocks the final path of platelets aggregation by preventing fibrinogen binding to platelets. This 26 amino acid fused hirudin peptide was artificially synthesized, purified and analysed.The study finally showed that the fused hirudin peptide had activities of antithrombin and antiplatelet. It is feasible to construct a hybrid multifunctional peptide that targets various components of haemostatic process.
