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[Isolation and culture of neural stem cells in injured region of compressive spinal cord injury in adult rat].

Abstract

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.

CONCLUSION

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.

Authors+Show Affiliations

Second Department of Orthopedics, Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an Shaanxi, 710004, P.R. China. yplyplypl1974@163.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

English Abstract
Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

19275093

Citation

Yang, Pinglin, et al. "[Isolation and Culture of Neural Stem Cells in Injured Region of Compressive Spinal Cord Injury in Adult Rat]." Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi = Zhongguo Xiufu Chongjian Waike Zazhi = Chinese Journal of Reparative and Reconstructive Surgery, vol. 23, no. 2, 2009, pp. 151-5.
Yang P, He X, Li H, et al. [Isolation and culture of neural stem cells in injured region of compressive spinal cord injury in adult rat]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(2):151-5.
Yang, P., He, X., Li, H., Lan, B., Wang, G., Liu, Y., & Li, Q. (2009). [Isolation and culture of neural stem cells in injured region of compressive spinal cord injury in adult rat]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi = Zhongguo Xiufu Chongjian Waike Zazhi = Chinese Journal of Reparative and Reconstructive Surgery, 23(2), 151-5.
Yang P, et al. [Isolation and Culture of Neural Stem Cells in Injured Region of Compressive Spinal Cord Injury in Adult Rat]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2009;23(2):151-5. PubMed PMID: 19275093.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Isolation and culture of neural stem cells in injured region of compressive spinal cord injury in adult rat]. AU - Yang,Pinglin, AU - He,Xijing, AU - Li,Haopeng, AU - Lan,Binshang, AU - Wang,Guoyu, AU - Liu,Yiheng, AU - Li,Qiang, PY - 2009/3/12/entrez PY - 2009/3/12/pubmed PY - 2009/6/13/medline SP - 151 EP - 5 JF - Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery JO - Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi VL - 23 IS - 2 N2 - 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. CONCLUSION: 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. SN - 1002-1892 UR - https://www.unboundmedicine.com/medline/citation/19275093/[Isolation_and_culture_of_neural_stem_cells_in_injured_region_of_compressive_spinal_cord_injury_in_adult_rat]_ L2 - https://medlineplus.gov/spinalcordinjuries.html DB - PRIME DP - Unbound Medicine ER -