血管组织工程种子细胞的建立及意义

摘要

目的:利用人Nanog基因转染血管组织工程的种子细胞(血管内皮细胞ECV304),提高其增殖能力,以在较短时间内获得大量种子细胞,从而为克服血管组织工程种子细胞来源的匮乏及组织工程血管的快速构建提供新途径.方法:制备含有人Nanog基因的复制缺陷型重组腺相关病毒血清2型病毒颗粒rAAV2-hNanog,用rAAV2-hNanog转染ECV304细胞,然后采用RT-PCR检测hNanog基因在转染的ECV304细胞中的表达.再用MTT、免疫荧光及激光共聚焦技术检测hNanog基因对血管内皮细胞ECV304生长的影响.结果:(1)rAAV2-hNanog转染ECV304细胞后,RT-PCR检测表明hNanog基因能在血管内皮细胞ECV304中稳定表达;同时,血管内皮细胞在转染后的增殖能力也显著增强(P<0.05).光镜下观察发现,转染的血管内皮细胞的形态无明显改变.结论:转染hNanog基因后,血管内皮细胞的形态无显著变化,但其增殖能力明显增强.利用hNanog基因提高血管内皮细胞的增殖能力,能够克服血管组织工程种子细胞来源不足的瓶颈,为组织工程血管的快速构建及应用提供一种新的方法.%Objective:To promote the proliferation of seeding cells for tissue engineering blood vessel,we transfected human Nanog gene into vascular endothelial cell mediated by recombinant adenovirus - associated virus Ⅱ type ( rAAV2 ). By this means, we can obtain enormous engineering cells for tissue engineering blood vessel in relatively short time and facilitate the construction of artificial vessel. Methods:To prepare the recombinant adenovirus -associated virus Ⅱ type harboring human Nanog gene (rAAV2 -hNanog) ,then ,we detected the expression of human Nanog in ECV304 cell by RT -PCR after transfection. Finally, we tested the effect of human Nanog on ECV304 cell line using tetrazolium salt colorimetry ( MTT), confocal laser scanning microscope( CLSM ). Results:The human Nanog can be expressed in engineering cell ECV304. The proliferation activity of transfected seeding cell was significantly improved( P ＜ 0.05 ). Meanwhile, no obvious morphology changes could be seen under inverted microscope in the seeding cell transfected by rAAV2 -hNanog compared with control cells. Conclusion :The proliferation activity of transfected engineering cell ECV304 was enhanced without significant morphological changes in the transfected cell. In brief, the proliferation of engineering cell ECV304 modified by human Nanog gene was obviously accelerated. This new method will overcome the insufficiency of seeding cells for tissue blood vessel.