关键词: 羊膜上皮细胞, 碱性成纤维细胞生长因子, 慢病毒, 基因转染, 免疫荧光细胞化学, 酶联免疫吸附法, 大鼠

Abstract: Objective To construct rat amniotic epithelial cells (AECs) modified with human basic fibroblast growth factor (bFGF) gene that combined with nerve growth factor (NGF) secreting peptide utilizing lentivirus vector, by which bFGF could be stably synthesized and secreted. Methods Amniotic epithelial cells were collected from the term rats by trypsin-treating process, and subcultured AECs were identified by immunocytochemistry and RT-PCR methods. Lentiviral vector with human bFGF gene was constructed, and lentivirus packaging was performed after DNA sequencing. Afterwards, AECs were infected with viral supernatant, then selected with blasticidin. Furthermore, the selected cells were detected for bFGF expression and secretion by immunocytochemistry, RT-PCR and ELISA methods. Thereafter，growth activity of AECs chimaera was investigated, and the biological functions of secreted bFGF was evaluated by culturing PC12 cells with conditioned medium of AECs chimaera. Results Immunocytochemical staining and RT-PCR showed that the rat AECs expressed epithelialspecific markers CK-19, neural cell markers nestin and GFAP, as well as pluripotent cells markers SSEA-4, Oct-4, Nanog, Sox2 and vimentin. The AECs chimaera sustainable expressing bFGF were obtained after infection and screening processes. Immunocytochemistry staining and RT-PCR showed that most of the AECs chimaera expressed bFGF, compared with the nonexpression before transfection. ELISA results implicated that AECs chimaera were able to secrete bFGF peptide. AECs chimaera survived in the screening procedure grew much faster compared with common AECs. The conditional culture medium of AECs chimaera could stimulate PC12 cells growth and neurites development, as compared with AECs group. Conclusion bFGF gene could be transferred into rat AECs successfully by lentiviral vector, and the AECs chimaera could express and secret bFGF afterwards, indicating robust growth activity and significant neurotroph

Key words: Amniotic epithelial cell, Basic fibroblast growth factor, Lentivirus, Gene transfection, Fluorescence immunocytochemistry, ELISA, Rat