Effect Of Controlled Release Of Cytokines From Collagen Gel On Neural Stem Cells

Neurons can not regenerate after central nervous system injury,and the movement,sensation and sphincter function was destroyed below the damaged segment.The current treatment can only delay the spinal cord injury,but it can not reverse the damaged nerve tissue.However,there is not an effective solution to better address this focus until recently the identification of stem cells in the adult human brain has opened up a new avenues in neuroregeneration.NSCs could proliferation and differentiated into neurons,astrocytes and oligondendrocytes.However,the effectiveness of NSCs transplantation in the spinal cord injury is limited,because the cavity remains at the lesion site prevents the axonal growth of the injured spinal cord and the migration of transplanted neural stem cells.Nowadays,the neural tissue engineering technology is a research hotspot.Here,we isolated and cultured the neural stem cells by nerusphere assay,NSCs cultured in different time were induced to differentiated and to detect the change of differentiation properties.Then,NSCs were seeded into a three-dimensional collagen gel scaffold,and we observed the compatibility of neural stem cells in collagen gel.At last,we formed the collagen gel loaded with cytokine,and tested its effect on the NSCs.The study consists of three parts.In part I,NSCs were isolated from rat'embryonic cerebral cortex and cultured in serum-free medium.Immunocytochemical staining was used to examine NSCs and its differentiated cells marks.NSCs grown as free-floating neurospheres in the serum-free conditions,but spontaneous fusion occurred among some neurospheres.Immunocytochemical staining showed these neruospheres positive expression of nestin and NSCs could differentiated into neurons,astrocytes and oligondendrocytes,and theproportion of astrocytes was the highest.In part II,NSCs were seeded into a three-dimensional collagen gel scaffold,with suspension cultured NSCs being used as a control group.Neural stem cells,which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor,actively expanded and formed neurospheres in both culture groups.In serum-free medium conditions,the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group.Immunofluorescence staining showed that neurospheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker ?III-tubulin,the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2',3'-cyclic nucleotide 3'-phosphodiesterase.Compared with neurospheres cultured in suspension,the differentiation potential of neural stem cells cultured in collagen gels increased,with the formation of neurons at an early stage.In part III,We formed collagen gel loaded with cytokine(BDNF and/or NT-3),With three groups of collagen gel,BDNF/collagen gel,and NT-3/collagen gel as controls,BDNF and NT-3 were tested in the BDNF-NT-3/collagen gel,BDNF/collagen gel and NT-3/collagen gel groups at different time points.The enzyme-linked immunosorbent assay results showed that BDNF and NT-3 were steadily released from collagen gels for 10 days.The cell viability test showed that BDNF-NT-3/collagen gel supported the survival and proliferation of NSCs.The results also showed that the length of processes was markedly longer and differentiation percentage from NSCs into neurons was much higher in the BDNF-NT-3/collagen gel group than those in the collagen gel,BDNF/collagen gel,and NT-3/ collagen gel groups.The proportion of neurons was the highest in BDNF-NT-3/collagen gel group.In the BDNF-NT-3/collagen gel and BDNF-collage gel groups,the differentiated cell phenotypes in quantities showed the following order: neurons > astrocytes > oligodendrocytes,whereas for the other two groups,the differentiated cell phenotypes in quantities showed the following order: astrocytes > neurons > oligodendrocytes.In conclusion,in serum-free medium containing growth factors,cerebral cortex neural stem cells could be isolated and cultured by using neurosphere assay.Neural stem cells have the properties of self-renewal,differentiating multipotentiality,and the ability to proliferate.Collagen gel has a good biocompatibility with NSCs.Collgan gel not only can as a three dimensional scaffold for the adhesion and migration of NSCs,as well as for the process extension,but also serve as a reservoir for neurotrophic factors to prolong its half-life and promote the function of neurotrophic factors which could significantly improve the ability of NSCs proliferation and differentiation,and the combination use of multiple cytokines have more effect on neural stem cells.