[Isolation and culture of neural stem cells in injured region of compressive spinal cord injury in adult rat].
OBJECTIVE
To investigate the division, proliferation and differentiation abilities of nestin+/GFAP+ cell after spinal cord injury and to identify whether it has the characteristic of neural stem cells (NSCs).


METHODS
Twelve male SD rats, aged 8 weeks and weighing 200-250 g, were randomized into 2 groups (n=6 per group): model group in which the spinal cord injury model was established by aneurysm clip compression method, and control group in which no processing was conducted. At 5 days after modeling, T8 spinal cord segment of rats in each group were obtained and the gray and the white substance of spinal cord outside the ependymal region around central tube were isolated to prepare single cell suspension. Serum-free NSCs culture medium was adopted to culture and serum NSCs culture medium was applied to induce differentiation. Immunohistochemistry detection and flow cytometry were applied to observe and analyze the type of cells and their capability of division, proliferation and differentiation.


RESULTS
At 3-7 days after injury, the model group witnessed a plenty of nestin+/GFAP+ cells in the single cell suspension, while the control group witnessed few. Cell count of the model and the control group was 5.15 +/- 0.71 and 1.12 +/- 0.38, respectively, indicating there was a significant difference between two groups (P < 0.01). Concerning cell cycle, the proportion of S-phase cell and proliferation index of the model group (15.49% +/- 3.04%, 15.88% +/- 2.56%) were obviously higher than those of the control group (5.84% +/- 0.28%, 6.47% +/- 0.61%), indicating there were significant differences between two groups (P < 0.01). In the model group, primary cells gradually formed three-dimensional cell clone spheres, which were small in size, smooth in margin, protruding in center and positive for nestin immunofluorescence staining, and large amounts of cell clone spheres were harvested after multiple passages. While in the control group, no obvious cell clone spheres was observed in the primary and passage culture of single cell suspension. At 5 days after induced differentiation of cloned spheres in the model group, immunofluorescence staining showed there were a number of galactocerebroside (GaLC) -nestin+ cells; at 5-7 days, there were abundance of beta-tubulin III-nestin+ and GFAP-nestin+ cells; and at 5-14 days, GaLC+ oligodendrocyte, beta-tubulin II+ neuron and GalC+ cell body and protruding were observed.


CONCLUSIONS
Nestin+/GFAP+ cells obtained by isolating the gray and the white substance of spinal cord outside the ependymal region around central tube after compressive spinal cord injury in adult rat has the ability of self-renewal and the potential of multi-polarization and may be a renewable source of NSCs in the central nervous system.
Yang
Pinglin
P
Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China. yplyplypl1974@163.com
He
Xijing
X
Li
Haopeng
H
Lan
Binshang
B
Wang
Guoyu
G
Liu
Yiheng
Y
Li
Qiang
Q
D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D008297:Male; D009416:Nerve Regeneration; D009474:Neurons / Q000166:cytology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013119:Spinal Cord Injuries; D013234:Stem Cells / Q000166:cytology*
D005904:Glial Fibrillary Acidic Protein