ABSTRACT
Objective To seek an optimal method for the separation,culture of mouse embryonic germ cells (EGCs) in vitro,and to observe the influence of Activated Schwann cells (ASCs)-derived neurotrophins on the differentiation capability of mouse EGCs into neurogenic cells.Methods The gonadal ridges and a few abdominal tissues of the 11-day postcoitum (dpc) mouse embryos were isolated and disaggregated by 0.125％ trypsin-0.02％ EDTA,followed by culture of the mouse EGCs on mouse embryonic fibroblast (MEF) feeders.Monoclonal formation of the mouse EGCs was observed,and the staining of stage specificity embryo antigen-1 (SSEA-1),alkaline phosphatase (AKP),periodic acid-Schiff staining (PAS) were applied to identify the mouse EGCs.Two groups were divided as followed:mouse EGCs+basic medium (control group) and mouse EGCs+ASCs (experimental group).Immunofluorescence (NeuN,MBP,GFAP)analysis was used to evaluate the neurogenic differentiation of mouse EGCs and then to calculate the statistical positive rates of cell staining.All experimental results were analyzed statistically.Results (1) Identification ofmouse EGCs:Mouse EGCs were characterized by a dome-shaped colony containing a large nucleus and a relatively small amount of cytoplasm.All mouse EGCs were positive staining of SSEA-1,AKP,and PAS;(2)The neural induction of mouse EGCs:After one week induction,there were few round or oval cells with long axon-like processes migrating from the edge of the EGCs clones.3 weeks later,the neurogenic-like cells increased quickly.The results of immunofluorescence (NeuN,MBP,GFAP)staining demonstrated that mouse EGCs could differentiate into neurogenic cells under the influence of ASCs.The positive rate of cell staining was significant.Conclusion In this study,a simple,economical method was applied to successfully separate the mouse EGCs in vitro; mouse EGCs can differentiate into neurogenic cells under the influence of ASCs-derived neurotrophins.
ABSTRACT
BACKGROUND:Schwann cells can secrete various neurotrophic factors,and promote functional recovery of injured spinal cord.However,xenogenic Schwann cells transplantation may induce autoimmune response.Moreover,local transplantation results in secondary injury.Vein transplantation may reached injury site passing the blood spinal cord barrier,but the treatment concentration is not effective.OBJECTIVE:To investigate the therapeutic effects of transplantation of autologous activated Schwann Cells(AASCs)via subarachnoid space on spinal cord injury(SCI)in rats.METHODS:A total of 66 rats were used to establish SCI models,and the model rats were randomly divided into 3 groups.The unilateral saphenous nerves of rats were ligated directly for 1 week to activate Schwann cells,but inactivated and model control groups were not subjected to nerve ligation.1 cm nerve was excised from distal end of each group,and Schwann cells were isolated and cultured by tissue mass method.The AASCs,autologous Schwann cells(ASCs)were injected with corresponding Hoechst33342-labeted SCs suspension,but the model control group was injected with DMEM injection.The basso beattie bresnahan(BBB)score and footprint analysis,as well as HE and GFAP immunohistochemistry staining were performed to evaluate functional recovery of rat hind limbs.RESULTS AND CONCLUSION:On 4 weeks after injury,BBB scores of AASCs were significantly superior to the other groups (P＜0.05).Two weeks after transplantation,some SCs migrated to injured spinal cord.Compared with ASCs group,the center distance of forward and hind feet and extorsion angle of the third toe of hind limb were significantly reduced in the AASCs group at 5 weeks(P＜0.05),the glial scar area was significantly decreased at 13 weeks(P＜0.05),and the cavity area of injured region was signiflcentJy diminished(P＜0.05).Results show that AASCs transplantation via subarachnoid space promoted functional recovery after SCI in rats.
ABSTRACT
BACKGROUND: Emerging studies have focused on cell transplantation. Schwann cells (SCs) can secrete various neurotrophic factors and improve local environment around injury. Plenty of documents have demonstrated that SCs could promote functional recovery following spinal injury. Many transplanting methods are available for treating spinal cord injury, and the intravenous cell transplantation is profitable for easy operation and avoidance of additional trauma. OBJECTIVE: To investigate the effects of intravenous transplantation of SCs on spinal cord injury in rats. METHODS: The bilateral sciatic nerves of Wistar rats were separated in vitro, cultured by tissue clot method, identified by S-100 and labeled by Hoechst33342. Sixty rat models with T10 spinal cord injury were prepared using impactor model- II type weight drop apparatus. Then the injured rats were randomly divided into 3 groups: blank control, DMEM control and SCs transplantation groups. No treatment was performed in the blank control group. Totally 1 mL DMEM and or SCs was injected into rats of DMEM control and SCs transplantation groups by tail vein respectively. Basso Beattie Bresnahan (B6B) scores were performed at 1 day before and 1, 3 days, 1 week and weekly after operation. The migration of transplanted SCs was observed at 2 weeks and 4 after transplantation. The expressions of glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE) were detected by haematoxylin-eosin staining and immune-fluorescence staining.RESULTS AND CONCLUSION: The purity of SCs reached 95%. Hoechst33342 positive cells were observed throughout the injured and the nearby region of spinal cord at 1, 2, and 4 weeks after transplantation. The statistical difference of BBB score among the SCs transplantation, blank control, and the DMEM control groups displayed at 4 weeks after transplantation (P < 0.05), and the BBB scores of the SCs transplantation were higher than other groups. Haematoxylin-eosin staining showed the cavity formed in each group at 8 weeks after transplantation, but the area of SCs transplantation was smaller than that of the blank control and DMEM control groups. The immunofluorescence staining indicated that the expression of GFAP were more intense in the blank control group and DMEM control than SCs transplantation (P < 0.05), while the expression of NSE was more intense in SCs transplantation than other groups (P< 0.05). It implied that intravenous transplantation of SCs promotes regeneration of axon and improves neurological functions after spinal cord injury in rats.