Study of brain-derived neurotrophic factor gene transgenic neural stem cells in the rat retina.
BACKGROUND
Neural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas.


METHODS
NSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively.


RESULTS
NSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation.


CONCLUSIONS
The seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.
Zhou
Xue-mei
XM
Department of Ophthalmology, Second Affiliated Hospital of Harbin Medical University, Heilongjiang, China.
Yuan
Hui-ping
HP
Wu
Dong-lai
DL
Zhou
Xin-rong
XR
Sun
Da-wei
DW
Li
Hong-yi
HY
Shao
Zheng-bo
ZB
D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D009474:Neurons / Q000166:cytology*; D051381:Rats; D012160:Retina / Q000166:cytology* / Q000378:metabolism*; D033581:Stem Cell Transplantation
D019208:Brain-Derived Neurotrophic Factor