| ObjectivesTo establish methods of isolating and culturing the epidermal neuralcrest stem cells (EPI-NCSCs) from green fluorescent protein (GFP)transgenic rats, and to lay the foundation for tracking transplanted cell ofspinal cord injury and repair. To observe the effect of EPI-NCSCs on ratswith spinal cord contusion; To explore the mechanism of repair spinal cordinjury using transplantation of EPI-NCSCs by detecting the expression ofbrain-derived neurotrophic factor(BDNF)、nerve growth factor(NGF)、glialcell line-derived neurotrophic factor(GDNF)、 Nogo-A in spinal cordcontusion of rats.MethodsThe bulge of GFP rat whisker hair follicle were dissected, the bulgeswere then rolled out of capsule and placed into collagen-coated cultureplates. Three days post-explantation, EPI-NCSCs were observedemigrating from the bulge explants, then these bulges were removed and the adhering cells were resuspended by trypsinization, and placed in freshcollagen-coated culture plates. The EPI-NCSCs were identified by theimmunocytochemistry of Sox10and Nestin, and its purity was calculatedaccording to the positive rate.The rat models of spinal cord injury (SCI) were made by NYU-Ⅱimpactor (10g25mm injuries force) at T10level. Then60SD rats wererandomly divided into blank injury group (group A), mediumtransplantation group (group B), epidermal neural crest stem celltransplantation group (group C). The EPI-NCSCs were transplanted intothe injured region in the group C after1week of SCI. The group B used theDMEM/F12to replace EPI-NCSCs. Nothing was transplanted in blankinjury group. Then the locomotor function was appraised at1st week,2ndweek,3rd week,4th week,5th week and6th week after transplantation byBasso-Beattie-Breanahan locomotor rating scale (BBB scale).3rats from each group, one week post-transplatation， thecorresponding15mm injuried spinal cord of T10were isolated from the rats.The expression level of BDNF、NGF of the three groups were detected byELISA.3rats from each group,3weeks post-transplatation，the corresponding15mm injuried spinal cord of T10were took out from the rats, and weremade into frozen section to detect expression of BDNF byimmunohistochemistry and immunofluoresence; the expression of Nogo-A was detected by immunofluoresence and Western blot; the expression levelof BDNF、NGF were also detected by ELISA.3rats from each group,6weeks post-transplatation，the corresponding15mm injuried spinal cord of T10were dissected from the rats, and weremade into frozen section, the expression of BDNF, GDNF and NGF weredetected by immunohistochemistry and immunofluoresence; the expressionof BDNF and Nogo-A were also detected by immunofluoresence andWestern blot; the expression level of BDNF、NGF were detected byELISA； the expression level of GDNF were detected by Real-timequantitative PCR.ResultsIt was found that the strong green fluorescence in the EPI-NCSCs fromGFP rats could be demonstrated under fluorescent microscope. Three dayspost-transplatation, the cells were observed emigrating from the blugeexplants onto the culture substratum, and then EPI-NCSCs appearedvariety typical morphology: rotundity, fusiform and trigon. More than90%cultured cells were identified to be EPI-NCSCs, which could expressSox10and Nestin.For all groups,2weeks after the transplantation, the rats of group Cdisplayed slight movement of one or two joints.4weeks after thetransplantation, the rats of group C were observed extensive movement ofthree joints.6weeks after the transplantation, the rats of group C displayed frequent to consistent weight-supported plantar steps. In contrast, the rats inthe other two groups could only sweep with no weight support and thepaws of the rats were the state of no weight support. The locomotorfunction of rats in the group C was significantly improved compared withthat in the other two groups. The results showed that the transplantation ofEPI-NCSCs could repair the locomotor function of SCI rats.One week after transplantation, there was no difference for theexpression level of BDNF and NGF in the all groups.3weeks after transplantation, the expression of the BDNF and NGF ingroup C was higher than that of group A and group B (P＜0.05) by ELISA;there was a small amount expression of BDNF in cell cytoplasm of graymatter and white matter of the spinal cord in group A and group B, butthere was higher expression of BDNF in group C in immunohistochemistrydetecting, the yellow masculine production was distributed throughout thetissue which was near the EPI-NCSCs in spinal cord injury; the number ofBDNF red masculine cells in group C was more than that of group A andgroup B, but the number of Nogo-A red masculine cells in group C wasless than that of group A and group B. It was also found that the expressionlevel of Nogo-A in group C was lower than the other two groups byWestern blot detecting, the difference was statistically significant(P＜0.05).6weeks after transplantation, the expression level of BDNF and NGF in the three groups were detected by ELISA. The expression of the BDNFand NGF in group C was sostenuto higher than that in group A and group B(P＜0.05); there was only slight expression of BDNF, GDNF, NGF in cellcytoplasm of gray matter and white matter of the spinal cord in group Aand B, but there was higher expression in group C; the number of BDNF,GDNF, NGF red masculine cells in group C was more than that of group Aand B, but the number of Nogo-A red masculine cells in group C was lessthan that of group A and B. It was also found that the expression level ofBDNF in group C was higher than the other two groups and the expressionlevel of Nogo-A in group C was lower than the other two groups byWestern blot, the difference was statistically significant(P＜0.05); theexpression of GDNFmRNA in group C was greatest in the three groups, thedifference was statistically significant(P＜0.05).Conclusion1. EPI-NCSCs from green fluorescent protein (GFP) transgenic ratscan express intensive, persistent green fluorescence. EPI-NCSCs of GFPcan be used as an ideal tool for the research of repair spinal cord injury bytransplantation of EPI-NCSCs.2. The motor function of rats can be enhanced by EPI-NCSCstransplantation into contusion spinal cord.3. The repair the spinal code injury by transplantation of EPI-NCSCsis related to the increasing of BDNF, NGF and GDNF expression and reducing of Nogo-A expression in injury spinal cord. |
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