Biological characteristics of rat neural stem cells transfected with human brain-derived neurotrophic factor and green fluorescent protein genes
10.3760/cma.j.issn.1671-8925.2009.08.008
- VernacularTitle:hBDNF-GFP基因转染神经干细胞后hBDNF的表达及生物学特性研究
- Author:
Dong-Fu FENG
1
;
Yong LIU
;
Er-Tao CHEN
;
Yang WANG
;
Tian-Jin LIU
Author Information
1. 上海交通大学医学院附属第三人民医院
- Keywords:
Stem cells;
Brain derived neurotrophic factor;
Green fluorescent protein;
Gene transfection
- From:
Chinese Journal of Neuromedicine
2009;8(8):785-789
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the expression of human brain-derived neurotrophic factor (hBDNF) and green fluorescent protein (GFP) in hBDNF-GFP gene-transfected rat neural stem cells (NSCs) and the changes in the biological characteristics of the transfected cells. Methods NSCs were transfected with a lentiviral vector carrying hBDNF and GFP genes (hBDNF-GFP-NSCs) or GFP gene only (GFP-NSCs), with normal NSCs as the control. The expression levels of hBDNF mRNA and hBDNF protein in all the 3 groups were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively. Enzyme-linked immunosorbent assay (ELISA) was performed to detect hBDNF level in the cell culture medium before and after hBDNF-GFP gene transfection. Dorsal root ganglion (DRG) neurons and NSCs were cultured with the supernatants of the transfected NSCs and normal NSCs, and the growth status of the DRG neurons was observed and the proportion of NSCs differentiating into neurons determined. Results Compared with GFP-NSCs and normal NSCs, hBDNF-GFP-NSCs showed obvious hBDNF overexpression at both rnRNA and protein levels 7 days after the transfection, hBDNF content in the supematant of hBDNF-GFP-NSCs culture increased significantly with time and peaked 5 days after the transfecfion (P<0.05). Four days after culture in hBDNF-GFP-NSCs supernatant, the DRG neurons and adherent NSCs extended cells processes, and the ratio of the NSCs differentiating into neurons was higher in cells cultured in hBDNF-GFP-NSCs supematant than in those culture in GFP-NSCs and normal NSCs supematants. Conclusion Lentivitus can be used as the vector for hBDNF and GFP gene transfection into NSCs, and hBDNF-GFP gene-transfected NSCs maintain the basic characteristics of NSCs and are capable of stable expression and secretion of hBDNF and GFP.
