腺病毒载体携带标记基因EGFP转染人羊膜上皮细胞

发布时间：2018-07-10 12:44

本文选题：腺病毒科感染 + 绿色荧光蛋白质类　；参考：《中国组织工程研究》2017年21期

【摘要】：背景:人羊膜上皮细胞具有干细胞的某些性能,可诱导分化为角膜样上皮细胞,但在体外培养时无法示踪,不能有效地监测其细胞转归。目的:探讨利用腺病毒载体将标记基因EGFP转染入人羊膜上皮细胞的可行性及感染效率。方法:pD C316-mC MV-EGFP腺病毒包装后,腺病毒载体携带标记基因EGFP转染体外培养的人羊膜上皮细胞,并扩增培养,观察转染后人羊膜上皮细胞与未转染人羊膜上皮细胞的形态差异,并在荧光显微镜下观察转染基因的表达,用流式细胞仪检测转染细胞的细胞周期及EGFP阳性表达率。结果与结论:(1)多数转染细胞筛选出的抗性克隆以及培养的克隆细胞与正常体外培养的人羊膜上皮细胞在形态上无显著差异;(2)瞬时转染后48 h的人羊膜上皮细胞中增强型绿色荧光蛋白阳性表达率最高达99.01%;(3)转染后的人羊膜上皮细胞的细胞周期有所减慢,说明利用腺病毒载体能将标记基因EGFP转染入人羊膜上皮细胞,可更直观地观察到人羊膜上皮细胞在体外的进一步转归。
[Abstract]:Background: human amniotic epithelial cells (HMECs) have some properties of stem cells, which can induce and differentiate into keratoid epithelial cells, but can not be traced in vitro and can not effectively monitor the outcome of human amniotic epithelial cells. Aim: to investigate the feasibility and infection efficiency of transfection of labeled gene EGFP into human amniotic epithelial cells by adenovirus vector. Methods Adenoviral vector carrying marker gene EGFP was transfected into human amniotic epithelial cells cultured in vitro, and the morphological differences between human amniotic epithelial cells and untransfected human amniotic epithelial cells were observed. The expression of transfection gene was observed under fluorescence microscope. The cell cycle and EGFP positive expression rate of transfected cells were detected by flow cytometry. Results and conclusions: (1) there was no significant difference in morphology between the majority of transfected cells and the cultured human amniotic epithelial cells in vitro, and (2) 48 h after transient transfection of human amniotic epithelial cells, there was no significant difference in morphology between the cloned cells and normal human amniotic epithelial cells. The highest positive expression rate of enhanced green fluorescent protein was 99.01. (3) the cell cycle of transfected human amniotic epithelial cells slowed down. The results showed that EGFP could be transfected into human amniotic epithelial cells by adenovirus vector, and the further development of human amniotic epithelial cells in vitro could be observed more intuitively.
【作者单位】：深圳市宝安人民医院眼科;暨南大学附属第一医院眼科;
【基金】：国家自然科学基金项目(30872808,81100637) 广东省医学科学技术研究基金项目(201512902216517) 广东省科技计划项目(2015A020212026) 广东省中医药局科研项目(20162044) 深圳市科技计划基金项目(JCYJ20140414105820176,CXZZ20140418182638764)~~
【分类号】：R3416

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