转基因内皮细胞的构建及其在复合血管上种植的初步研究

The Construction of VEGF Transferred HUVEC and Seeding on the Luminal Surface of Biosynthetic Vascular Grafts

目的:为了解决小口径(直径7mm以下)人造血管术后通畅率低下的问题,利用生物-人工复合血管,结合分子生物学手段,将转入血管内皮生长因子(VEGF165)基因的人脐静脉内皮细胞(HUVEC)种植于复合血管内腔面,为解决此临床难题提供新的思路和手段。材料方法:构建VEGF165真核表达质粒pCI-neo-VEGF165,获得正确的质粒用于转染HUVEC。从中科院购买HUVEC(代号:ECV-304),用脂质体介导法将pCI-neo-VEGF165转入细胞,经G418筛选后,计数阳性克隆,以比较脂质体和质粒在不同比例情况下转染效率的高低。实验组按质粒和脂质体比例的不同分为4组:1μg/3μl;2μg/6μl;2μg/8μl;3μg/12μl。ELISA检测瞬时转染后HUVEC培养上清中VEGF的表达。再将VEGF165基因瞬时转入HUVEC,利用自己设计的旋转培养装置,将细胞均匀地粘附、种植于复合血管内腔面。复合血管是人工材料(聚酯)和生物材料(成纤维细胞长入,胶原形成)有机结合而成,是将包有聚酯网的硅胶棒埋入绵羊背部皮下组织,3个月后取出,抽去硅胶棒而得到的。结果:脂质体转染效率以2μg/8μl组阳性克隆数最多;ELISA测定瞬时转染后HUVEC培养上清中VEGF的表达量为532·5±43·1pg/ml。使用旋转培养装置的细胞均匀地粘附于复合血管,未使用者细胞积聚于血管腔面下部;转入和未转入VEGF165基因的HUVEC在复合血管内腔面覆盖面积百分比分别为69·9%±3·5%、43·7%±2·5%,有明显改善。结论:转入VEGF后可促进HUVEC在复合血管上生长,旋转培养装置有利于细胞的均匀粘附。

Purpose： To solve the obstruction of small-bore artificial grafts after operation, we seeded HUVEC transferred with VEGF165 on the luminal surface of biosynthetic vascular graft （a new-type biological-artificial graft） to exploit a novel method of fabricating bio-artificial grafts with small diameters. Material and Method： First, to construct the eukaryotic expression plasmid with VEGF165 inserted. We cut the VEGF fragment from pCR2.1-VEGF165 with restriction enzyme EcoR I, and linked it with eukaryotic mammalian expression vector pCI-neo. As a result, we got a clone named pCI-neo-VEGF165, which experienced both EcoR I and Nde I digestion appraisals and was proved to have VEGF165 existing in correct direction. Second, to transfer pCI-neo-VEGF165 into HUVEC （ECV-304, purchased from Chinese Academic Institution of Sciences） by liposome-mediated method. So we optimized the transfection condition for afterword experiments and obtained the gene transferred HUVEC for next step utilization. Third, to seed VEGF165 transferred HUVEC on the luminal of the biosynthetic vascular grafts with fibronectin in the self-designed rotary culture equipment. Result： The optimal ratio of liposome and plasmid was 2μg/8μl for the transfection of HUVEC. The coverage ratio of gene transferred and untransferred HUVEC was 69.9%±3.5%, 43.7%±2.5% respectively. Conclusion： The proliferation of HUVEC on the biosynthetic vascular grafts was obviously improved by transferring VEGF165 to HUVEC.