Establishment of embryonic neural stem cell clone in rats following ordinal treatment of basic fibroblast growth factor and epidermal growth factor / 中国组织工程研究
Chinese Journal of Tissue Engineering Research
; (53)2006.
Artigo
em Chinês
| WPRIM (Pacífico Ocidental)
| ID: wpr-593391
Biblioteca responsável:
WPRO
ABSTRACT
BACKGROUND:
The effective method to obtain neural stem cells through in vitro culture that deserves to pay more attention in experimental studies of stem cells.OBJECTIVE:
To investigate the effective methods to culture neural stem cells in vitro using culture medium containing different growth factors.DESIGN:
Single sample observation.SETTING:
Laboratory of Cell Biology, College of Life Science, Sichuan University.MATERIALS Ten SD rats which were pregnant for 14 days, DMEM/F12 11, basic fibroblasts growth factors, epidermal growth factor; nestin antibody IgG , β- microtubule protein Ⅲ antibody IgG , glial fibrillary acidic protein antibody IgG, biotin labeled second antibody and third antibody and fluorescinisothiocyate (FITC)-labeled second antibody were used in this experiment.METHODS:
This experiment was carried out at the Laboratory of Cell Biology, College of Life Science, Sichuan University from 1999 to 2001. ① Brain tissue was taken out from the procerebrum of fetal rat, then primary neural stem cell clone was obtained through enzymatic digestion, mechanical treatment, centrifugation and culture. ② Neural stem cells were inoculated and cultured at 2×108 L-1 and divided into 4 groups with 6 bottles of cells in each group. DMEM/F12 (11 )culture medium containing 0.1 volume fraction of fetal bovine serum was added , serving as DMEM/F12 group; culture medium containing 20 μg/L basic fibroblast growth factor was added , serving as basic fibroblast growth factor group; culture medium containing 30 μg/L epidermal growth factor was added , serving as epidermal growth factor group; Culture medium containing basic fibroblast growth factors was added to culture for 2 hours, then culture medium containing epidermal growth factors was used for further culture, serving as basic fibroblast growth factor+ epidermal growth factor group. Culture flask was change after 24-hour culture; another 14 days later, primary clone number was counted under the microscope, and expressions of specially labeled albumen nidogen of stem cells were detected with immunohistochemistry.MAIN OUTCOMEMEASURES:
① Primary clone of neural stem cells.② Clone culture result of unicell. ③ Induction and differentiation results.RESULTS:
① The cells isolated from the brain of fetal rats possess the ability to consecutively passage and form clone , and immunofluorescent staining showed cell nidogen expression positive in the cell sphere. ②Clone rate of neural stem cells was the highest (0.630%)using ordinal culture of basic fibroblast growth factor and epidermal growth factor. ③ The cultured neural stem cell clone can be induced and differentiated neurons and glinl cells.CONCLUSION:
① The results proved that the cultured and isolated cells are neural stem cells. ②Ordinal treatment of basic fibroblast growth factor and epidermal growth factor is an effective method to obtain neural stem cells.
Texto completo:
Disponível
Base de dados:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Chinese Journal of Tissue Engineering Research
Ano de publicação:
2006
Tipo de documento:
Artigo