Cloning of the eukaryotic expression vector with nerve growth factor in rats and its effects on proliferation and differentiation of mesencephal neural stem cells of fetal rats.
The eukaryotic expression vector containing full-length cDNA sequence of rate nerve growth factor (NGF) beta subunit was constructed and its effects on proliferation and differentiation of neural stem cells were observed. By using PCR, full-length cDNA sequence of NGF beta subunit in rats was cloned and ligated into the eukaryotic expression vector pEGFP-N1-NGF. The recombinant plasmid pEGFP-N1-NGF was transfected into the mesencephal neural stem cells of embryonic rats by Lipofectamin and transiently expressed. MTT method was used to determine the effects of NGF on proliferation of neural stem cells, and under phase-contrast microscopy, the effects of NGF on growth of nervous processes following differentiation of neural stem cells were observed. Sequence analysis indicated that the cloned full-length cDNA sequence of rat NGF beta was identical to that of published sequence encoding NGF in gene GeneBank. The transfection of recombinant plasmid pEGFP-N1-NGF into mesencephal neural stem cells of embryonic rats could obviously promote proliferation of neural stem cells and facilitate the growth of neural stem cells-derived nerve cells. It was suggested that neural stem cells could be used as a vehicle of gene transfer, and the expression of NGF beta subunit in the neural stem cells could promote the growth of nerve cells derived from neural stem cells.
Lin
Minhua
M
Department of Neurosurgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. lmh62265681@163.com
Yang
Lin
L
Fu
Rong
R
Zhao
Hongyang
H
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D053595:Embryonic Stem Cells / Q000166:cytology*; D005822:Genetic Vectors / Q000235:genetics; D008636:Mesencephalon / Q000166:cytology*; D020932:Nerve Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D058953:Neural Stem Cells / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D020932:Nerve Growth Factor