This work was supported by a grant from The National Natural Sciences Foundation of China (30200281).
根据抗 PTCA 或支架后再狭窄的基因治疗需要多基因治疗的特点,用基因重组技术构建了 hVEGF165 和嵌合水蛭肽 (fused hirudin , FH) 融合基因,并克隆到真核表达载体 pcDNA3.0 中,通过脂质体介导将 pcDNA3.0/hVEGF165 - FH 转染到人内皮细胞株 (ECV304) 中, RT-PCR 及蛋白质印迹证明融合基因 hVEGF165 - FH 在 ECV304 细胞中得到表达 ( 分子质量为 24 ku 左右 ). 通过体外活性检测——— MTT 法检测 hVEGF165 - FH 对 ECV304 细胞增殖的影响,通过体外血管生成分析 hVEGF165 - FH 对内皮细胞株 ECV304 增殖的影响 . 通过体外抗栓活性检测,表明表达产物具有促进内皮细胞株增殖及加快血管生成的作用,同时显著抑制了 ADP 诱导的血小板聚集率 (P < 0.05) 并显著延长 APTT 和 TT (P < 0.05) . 实验结果表明,融合基因在内皮细胞株中得到表达,表达的融合蛋白具有 hVEGF165 和嵌合水蛭肽 (FH) 的双重活性,这为以后的融合基因治疗再狭窄的动物实验打下了良好基础 .
In order to construct a fusion gene encoding hVEGF165 and fused hirudin(FH), which can not only accelerate endothelial cell proliferation but also inhibit thrombosis, the eukaryotic expression vector hVEGF165-FH/pcDNA3.0 was constructed which contained the hVEGF165 and fused hirudin(FH). Then, the constructed vector was transfected into endothelial cell strain(ECV304). The hVEGF165-FH fusion gene transcription and protein expression were examined by RT-PCR and Western blot. The activities of fusion hVEGF165-FH were identified by serials of endothelial cell proliferation tests and antithrombotic tests in vitro. The results showed that the fusion protein was successfully expressed and it had biological activities of both hVEGF165 and fused hirudin. This new fusion gene with multi-function might be a new weapon to prevent restenosis after percutaneous coronary intervention(PCI).
沈 雳,陈少萍,秦永文,蔡在龙. hVEGF165 和嵌合水蛭肽融合基因的构建、表达和活性检测[J].生物化学与生物物理进展,2005,32(11):1061-1068
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