Glial cell-derived neurotrophic factors combined with transforming growth factor-beta 1 for in vitro differentiation of neural stem cells from rat spinal cord / 中国组织工程研究

ABSTRACT

BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materialsDMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.