Study of brain-derived neurotrophic factor gene transgenic neural stem cells in the rat retina

• Published in: Chinese medical journal Vol. 122; no. 14; pp. 1642 - 1649
• Main Authors: Zhou, Xue-mei, Yuan, Hui-ping, Wu, Dong-lai, Zhou, Xin-rong, Sun, Da-wei, Li, Hong-yi, Shao, Zheng-bo
• Format: Journal Article
• Language: English
• Published: China Department of Ophthalmology,Second Affiliated Hospital of Harbin Medical University,Harbin,Heilongjiang 150086,China%Harbin Veterinary Research Institute,China Agricultural Academy of Science,Harbin,Heilongjiang 150001,China 20-07-2009
• Subjects: Animals; Brain-Derived Neurotrophic Factor - genetics; Brain-Derived Neurotrophic Factor - metabolism; Cell Differentiation - physiology; Cell Movement - physiology; Cells, Cultured; Embryo, Mammalian - cytology; Enzyme-Linked Immunosorbent Assay; Immunohistochemistry; Neurons - cytology; Rats; Retina - cytology; Retina - metabolism; Stem Cell Transplantation; 基因疗法; 神经因子; 神经干细胞; Heidelberg retina angiograph; brain-derived neurotrophic factor; immunohistochemistry; neural stem cells; transplantation
• ISSN: 0366-6999; 2542-5641
• DOI: 10.3760/cma.j.issn.0366-6999.2009.14.008

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 differentJatJon 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 Jmmunosorbent 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.