慢病毒介导碱性成纤维细胞生长因子在大鼠羊膜上皮细胞中的表达和分泌

Expression and secretion of basic fibroblast growth factor in rat amniotic epithelial cells introduced by lentiviral vector

目的探讨以慢病毒作为载体,将带有分泌肽的人碱性成纤维细胞生长因子(bFGF)基因片段转入大鼠羊膜上皮细胞(AECs)中,构建能稳定表达并分泌bFGF多肽的细胞载体。方法取妊娠晚期SD大鼠的羊膜组织进行AECs原代培养,用免疫荧光细胞化学和RT-PCR法鉴定;构建包含人神经生长因子(NGF)分泌肽-bFGF编码基因的慢病毒载体,并行慢病毒包装,用病毒上清对传代大鼠AECs进行感染并筛选,经免疫荧光细胞化学、RT-PCR及酶联免疫吸附法(ELISA)检测基因转染效果,体外培养观察基因转染后的细胞生长活性;用基因转染细胞的培养上清对PC12细胞进行培养,检测所分泌bFGF多肽的生物学活性。结果所培养的大鼠AECs经鉴定能表达上皮特异性标志物CK-19、神经类细胞标志物巢蛋白(nestin)、胶质纤维酸性蛋白(GFAP)以及细胞多能性标志分子SSEA-4、Oct-4、Nanog、Sox2、波形蛋白(vimentin)等;经感染并筛选后扩增培养的大鼠AECs在mRNA水平及多肽水平均能检测到目的基因bFGF的表达,筛选后的转染细胞生长活性较未转染细胞明显提高,其培养上清能明显促进PC12细胞的生长和分化。结论用慢病毒作为载体能成功将bFGF基因转染入大鼠AECs中,所建立的基因修饰AECs能表达并分泌bFGF多肽,具有较强的生长活性及神经营养活性。

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 Amniotie epithelial cells were collected from the term rats by trypsin-treating process, and subeultured 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 PCl2 cells with conditioned medium of AECs chimaera. Results Immunoeytoehemieal 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 ceils 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 euhure medium of AECs chimaera could stimulate PCl2 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 neurotrophic activity.