| Background:Traumatic SCIs represent a critical issue in clinical situation nowadays, and injured patients undergo severe physiological, psychological, and social failures. Even if slight recovery can be achieved with intensive training programs and treatments, this recuperation is slow and limited, and depends on the degree of injury. Despite many advances in the medical and surgical treatment of SCI patients, there are still no effective methods for the neurological deficits caused by SCI. Cell-based therapies could consequently provide additional expectations in the case of spinal cord lesions. In this study, we observed the effect that co-transplantation of bone mesenchymal stem cells (BMSCs) and olfactory mucosa neural stem cells (OM-NSCs) in the rat model of spinal cord injury. To the light of those observations, it appears that BMSCs and OM-NSCs could be used concomitantly:BMSCs could modulate inflammation, whereas OM-NSCs could then improve neuronal recovery and axonal sprouting.The first part:Isolation and identification of the rat olfactory mucosa neural stem cells (OM-NSCs) and bone mesenchymal stem cells (BMSCs)Objectives:To isolate rats BMSCs using horizontal shaking method. To investigate the purity and the differential ability of the extracted BMSCs. To establish the isolation and culture method of the OM-NSCs.Methods:Bone marrow was extracted from femur and tibia of healthy adult rats under sterile conditions. The original generation of the bone marrow stromal cells was shaken for 4 hours in a constant temperature shaking bed, abandon supernatant after subculture. The purity of BMSCs was identified by flow cytometry. The differentiation ability of the BMSCs was proved by osteogenic and adipogenic culture. Neural stem cells were extracted from olfactory mucosa of healthy adult rats under sterile conditions. The OM-NSCs was identified using immunohistochemical staining.Results:In the obtained BMSCs, the proportion of CD29 and CD90 positive cells were 99.1% and 9.19%, the proportion of CD31 and CD45 positive cells were 0% and 1.3%. After osteogenic and adipogenic culture, the BMSCs were able to differentiated to osteoblasts and adipocytes, resulting in calcium nodules and lipid droplets. The neural stem cells extracted from the olfactory mucosa were able to from typical neurospheres. Immunocytochemistry showed OM-NSCs labeled by P75, S100, Nestin and GFAP.Conclusion:The horizontal shaking method for BMSCs culture was stable and effective. This mothed could reduce the number of times of subculture, improve cell purity. The obtained cell had differentiation potential. The neural stem cells extracted from the olfactory mucosa were able to from typical neurospheres and could be identified as typical neural stem cells.The second part:In vitro Co-culture of bone mesenchymal stem cells and olfactory mucosa neural stem cellsObjectives:To investigate the secretion of nerve related neurotrophic factors and anti-apoptotic ability to protect neurons of BMSCs and OM-NSCs cultured in a direct co-culture system. To investigate the interaction of BMSCs and OM-NSCs in a indirect co-culture system.Methods:The content of NT-3 and NGF secreted by BMSCs and OM-NSCs in monoculture and direct co-culture system was determined in vitro. The expression of Nestin、 MAP-2、GFAP、Tuj1、NSF-1、MMP-2、MMP-9、MMP-13 in the BMSCs and OM-NSCs in monoculture and indirect co-culture system was determined by fluorescent quantitative PCR. Fetal rat neurons was extracted and stimulated by inflammatory cytokine (IL-1β). The stimulated neurons were indirect co-cultured with BMSCs, OM-NSCs and BMSCs mixed with OM-NSCs. The neuronal apoptosis rate was detected by TUNNEL and MAP-2 immunofluorescence assay.Results:The content of NGF was the highest and NT-3 was the lowest in culture medium of mono BMSCs. The content of NGF was the lowest and NT-3 was the highest in culture medium of mono OM-NSCs. The content of NGF an NT-3 was in the middle in culture medium of direct co-culture of BMSCs and OM-NSCs. The expression of MMP-2、 MMP-9 and MMP-13 of BMSCs in indirect co-culture condition was increased and the expression of MAP-2、GFAP、Tuj1 was decreased. The expression of NSF-1 of OM-NSCs was decreased and the expression of Nestin. MAP-2、GFAP、Tuj1、MMP-2、 MMP-9 and MMP-13 was increased. All the stem cells could reduce the rate of apoptosis of the inflammatory cytokine stimulated neurons and the mixture of the two stem cells had the best effect.Conclusion:In the direct co-culture condition, the nerve related neurotrophic factors secreted by the two stem cells could be complementary each other. In the indirect co-culture condition, BMSCs could help OM-NSCs maintain sternness and OM-NSCs could promote BMSCs to differentiate to neural cells. OM-NSCs could prevent BMSCs to migrate and BMSCs would help OM-NSCs to migrate. The mixture of the two stem cells had the best effect to protect neurons from apoptosis by the inflammatory cytokine stimulation.The third part:Co-transplantation of BMSCs and OM-NSCs for repair of spinal cord injury in ratsObjectives:To investigate the effect of Co-transplantation of BMSCs and OM-NSCs for repair of spinal cord injury in rats, and compared with BMSCs or OECs alone transplantation.Methods:The rat spinal cord injury model was established by modified Allen method. Twenty rats were randomly divided into 4 groups:the control group, BMSCs group, OM-NSCs group and BMSCs+OM-NSCs group. BBB scale was evaluated before transplantation, Id,3d,7d and 14d after transplantation. The injured spinal cords were obtained at 14d for immunofluorescence staining assay.Results:BBB scale were 5.6±0.8,7.6±1.1,9.6±1.5 and 10.8±1.3 for the control group, BMSCs group, OM-NSCs group and BMSCs+OM-NSCs group respectively at 21d. The BBB scale all had significant improvement when compared pre-transplantation. HE stain also showed smaller liquify area and less vesicles. Immunofluorescence staining assay showed the transplanted BMSCs and OM-NSCs can migrate to the injury area.Conclusion:Co-transplantation of BMSCs and OM-NSCs showed better results for spinal cord injury in rats, and the transplanted BMSCs and OM-NSCs can migrate to the injury area. |
PDF Full Text Request