关键词: 骨骼肌来源的干细胞, 诱导分化, 神经干细胞

Abstract: Objective Skeletal muscle-derived stem cells are a population of cells including several different kinds of immature progenitors residing in the skeletal muscle tissues,such as pluripotent stem cells,side population cells,satellite cells and activated satellite cells-myoblasts.These precursor cells function in the maintenance and repair of adult skeletal muscle injury and regeneration.It has been demonstrated that mice myogenic cell lineage C2C12 can be induced to differentiate into neurogenic lineage by genetic modification.One kind of muscle-derived stem cells,myoblasts derived from adult rat skeletal muscles,has not been known to possess this neural conversion ability.As a type of cell population,muscle-derived stem cells can provide the sufficient cell source,which will benefit not only the muscular atrophy,but also the central nervous system neurodegenerative diseases such as Parkinson's disease.Because these cells can be obtained from the patient per se,they possess the advantage of clinical value.Therefore,in this study,the feasibility of neural differentiation of adult rat skeletal muscle-derived stem cells in vitro has been investigated.Methods Tissue was isolated from six-months old Sprague-Dawley rat gastrocnemius muscle.Cells were enzymatically dissociated by the addition of a solution of dispase,collagenase,and CaCl₂.The slurry,maintained at 37 ℃ for 45 min,was triturated every 15 min,and then passed through 75 μm nylon mesh.The filtrate was centrifugalized at 1 000 r/min to precipitate the dissociated cells,the pellet was resuspended in F10 medium,and the suspension was plated on type I collagen-coated dishes.During the first 4 days of the primary cultures,cells were enriched by continuous differential attachment technique.After culturing for 4～6 passages,cells were plated as a suspension in a medium optimized for neural stem cells growth which contained Neurobasal A supplemented with B27,basic fibroblast growth factor and epidermal growth factor for 7～10 d.The expression of myoblast-specific protein desmin,neural stem cell-specific protein nestin and neuron-specific protein βⅢ-tubulin、microtubule associated protein 2 were detected with immunocytochemical techniques,western blot and realtime-PCR.Results Stem cells isolated from adult skeletal muscle tissues proliferated rapidly after 4 passages,and survived for 6 months in vitro.The sequential adhesion method threw away many fibroblast cells mixed in the precursor cells culture system.After proliferation andpurification,over 95% of the muscle-derived stem cells expressed desmin and some of them expressed βⅢ-tubulin,but none of these cells expressed nestin or microtubule associated protein 2.After 7～10 d cultivation in the medium optimized for neural stem cells growth,muscle-derived stem cells formed neurosphere-like aggregates.Identified by immunocytochemical methods,western blot and realtime-PCR,expression of nestin increased significantly and that of desmin declined sharply.βⅢ-tubulin expression was slightly reduced and microtubule associated protein 2 expression was not detected after induction.Conclusion Muscle-derived stem cells were highly enriched through our isolation and cultivation system,and 95% were desmin-positive myoblasts.Through replacement of F10 medium with medium optimized for neural stem cells growth,muscle-derived stem cells could be induced to differentiate into neural stem cells after 7～10 d in vitro.The expression of myogenic markers declined gradually and that of neurogenic markers increased.This indicates that in our present culture system,muscle-derived stem cells could be induced to process the neural conversion progression.Next step,we will induce the primary adult stem cells derived from skeletal muscle tissues to differentiate into neurons by some factors,in order that the muscle-derived stem cells can be used as a kind of cell source for nervous system diseases especially neurodegenerative diseases treatment.

Key words: skeletal muscle-derived stem cells, induction and differentiation, neural stem cells