Measuring the expression of exogenous BDNF of NSCs after infection of supernatant from recombi-nant retrovirus pLXSN-BDNF
10.3760/cma.j.issn.0254-5101.2009.09.004
- VernacularTitle:pLXSN-BDNF体外转染神经干细胞及其外源基因的表达
- Author:
Xuemei ZHOU
;
Huiping YUAN
;
Donglai WU
;
Ya WANG
;
Zhuo WANG
- Publication Type:Journal Article
- Keywords:
Neural stem cells;
Purification;
Recombinant retrovirus;
Real-time fluorescent quan-titative PCR assay;
ELISA assay
- From:
Chinese Journal of Microbiology and Immunology
2009;29(9):786-791
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop neural stem cells(NSCs) which can stably express exogenous brain-derived neurotrophic factor(BDNF) in vitro. Methods NSCs from the subependymal zone of embry-onic day 14.5(E14.5) rat brain were purified by limiting dilution assay and then infected with supernatant of recombinant retrovirus pLXSN-BDNF and retrovirus pLXSN. The original copy numbers of exogenous gene templates from three groups NSCs(pLXSN-BDNF viral infection group, pLXSN viral infection group, control group) were detected by fluorescent quantitative PCR(FQ-PCR). ELISA assay was used for determining the protein contents of BDNF of supernatant from three groups NSCs for six days continually after seeded in 24-well plates in the same cell density. Results NSCs were purified successfully by limiting dilution assay.The original copy numbers of exogenous BDNF gene templates from pLXSN-BDNF viral infection group by FQ-PCR were (19.57±0.65) × 10~3 copies/μl, higher than those of another two groups(P < 0.05). The protein contents of BDNF of supernatant from NSCs of pLXSN-BDNF viral infection group was highest among three groups and compared with another two groups had statistical significance (P <0.05) . Conclusion The purified NSCs can be transduced exogenous BDNF successfully with supematant of recombinant retrovir-us pLXSN-BDNF which provide experimental evidences and laying foundations for further research of retinal transplantation and quantization investigation of gene therapy for optic nerve injury.
