Human amniotic epithelial cells transfected by enhanced green fluorescent protein gene mediated by adenovirus vector

doi:10.3969/j.issn.2095-4344.2017.21.017

Abstract

Abstract:

BACKGROUND: Human amniotic epithelial cells have some properties of stem cells, which can be induced to differentiate into corneal epithelial cells, but cannot be traced in vitro.
OBJECTIVE: To investigate the feasibility and infection efficiency of adenovirus vector carrying enhanced green fluorescent protein (EGFP) into the human amniotic epithelial cells.
METHODS: The adenovirus vectors carrying EGFP was transferred into human amniotic epithelial cells cultured in vitro. After cultured and amplified, the morphology difference between transfected and non-transfected human amniotic epithelial cells was observed. The transfected human amniotic epithelial cells were observed under fluorescence microscope, and the cell cycle and the expression rate of EGFP in transfected human amniotic epithelial cells were detected by flow cytometry.
RESULTS AND CONCLUSION: No obvious difference in the cell morphology was found between transfected human amniotic epithelial cells and normal human amniotic epithelial cells cultured in vitro. Flow cytometry analysis revealed that the EGFP positive rate was highest and reached up to 99.01% at 48 hours after transient transfection. The cell cycle of human amniotic epithelial cells transfected by the adenovirus vector was slowed a bit. To conclude, the adenovirus vector is a good medium of transfecting EGFP into human amniotic epithelial cells, and makes it more convenient to observe the further transformation of human amniotic epithelial cells in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adenoviridae Infections, Green Fluorescent Proteins, Amnion, Epithelial Cells, Cell Cycle, Tissue Engineering

CLC Number:

R394.2

Jin Ling, Liu Xiao-yong, Xu Wei, Hao Xiao-ning, Niu Jing-yi, Wang Yi-ting, Cao Duan-rong. Human amniotic epithelial cells transfected by enhanced green fluorescent protein gene mediated by adenovirus vector[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3382-3387.

Figures/Tables 1

2.1 人羊膜上皮细胞形态在接种12 h内细胞可下沉贴壁，24 h后细胞逐渐变大，折光性降低，整个细胞轮廓变暗，完全伸展的细胞呈扁平的卵圆形(图1A)；2 d后，进入指数生长期，细胞数量明显增多, 呈不规则的多角形，四五天后90%细胞融合成片，形成单层。传代后人羊膜上皮细胞能较快贴壁，接种4-6 h后即可贴壁并开始生长，呈多形性；接种24 h后细胞生长加速，细胞扁平变大(图1B)；三四天后细胞呈90%融合，呈典型的铺路石外观，呈多形性，与原代无明显区别。培养的人羊膜上皮细胞加入转染液12 h后，有少数细胞从培养板上脱落下来漂浮于培养液中，细胞间变得稀疏(图1C)，终止转染后细胞形态和生长特性逐渐恢复正常，终止转染后48 h，细胞大多数转染细胞均与正常细胞形态上无显著差异。 2.2 荧光显微镜下观察EGFP的阳性表达转染后12 h即可见发绿色荧光的人羊膜上皮细胞，但数量较少，强度较弱，每个低倍视野下可见0-2个细胞(图2A)；转染后24 h发绿色荧光的人羊膜上皮细胞逐渐增多、荧光强度增强(图2B)；转染后48 h发绿色荧光的人羊膜上皮细胞继续增多，几乎所有存活细胞均发出绿色荧光(图2C)；转染后72 h发绿色荧光的人羊膜上皮细胞有所减少，荧光强度开始减弱(图2D)；转染后96 h发绿色荧光的人羊膜上皮细胞继续减少，荧光强度继续减弱(图2E)。 2.3 流式细胞仪检测活细胞比例及人羊膜上皮细胞pDC316-mCMV-EGFP基因转染效率流式细胞仪检测结果显示：未转染组人羊膜上皮细胞中，绿色荧光蛋白阳性细胞数为0.14%(图3B)，瞬时转染后12，24，48，72，96 h的人羊膜上皮细胞均可见绿色荧光蛋白阳性表达，其中瞬时转染后48 h的人羊膜上皮细胞绿色荧光蛋白阳性表达率最高，达99.01%(图3C)，但随着体外培养时间的延长和传代次数的增加其荧光表达率逐渐下降。 2.4 流式细胞仪检测细胞周期转染后48 h的G0/G1期人羊膜上皮细胞占总细胞数的85.51%，G2/M期占7.48%，S期占7.02%，G2/G1为2.0%，未转染的人羊膜上皮细胞G0/G1期占总细胞数的78.21%，G2/M期占8.58%，S期占13.21%，与未转染细胞的细胞周期相比，转染后人羊膜上皮细胞G0/G1期细胞百分比大于未转染组。转染后人羊膜上皮细胞S期的细胞百分比小于未转染组，说明经腺病毒介导EGFP转染的人羊膜上皮细胞的细胞周期有所减慢(图4)。"

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