Establishment Of Spinal Cord Anterior Horn Motor Neurons And Skeletal Muscle Cells Co-culture System

Denervated laryngeal skeletal muscle regeneration and functional reconstruction is thefocus of attention in the clinical,the reconstruction of the neuromuscular junction and thefusion and differentiation of myoblast stem cells to repair damaged muscle fibers is the keyfor the regeneration and functional recovery of a denervated skeletal muscle.So in order tobetter study the regeneration mechanisms of the denervated skeletal muscle like laryngealmuscle, and provide new therapeutic strategies and theoretical basis for interfering the keymolecular events during neuromuscular regeneration,establish an appropriate model invitro is crucial.Anterior spinal horn motor neurons-skeletal muscle cells co-culture is oneof the key technology.Currently, most of the known neuromuscular co-culture model in vitro usingserum-containing medium, it provide a functional model system for the formation of theneuromuscular junction in vitro, however, the use of the serum increased unnecessaryvariability.Because of that the use of serum makes it impossible to describe and quantifythe minimum factors when repeating the co-culture system in vitro. Therefore, it was alsodesigned new serum-free culture techniques that with serum-free medium forneuromuscular co-culture to promote the formation of a functional neuromuscular junction.The source of the motor neurons and skeletal muscle cells are not the same, it becomefacilitated to analysis the process of vertebrate neuromuscular junction development whenusing embryonic neurons and skeletal muscle cells isolated in vitro. Copy tissue-typeinteractions between cells is often difficult to achieve the effect of separation of embryonicstem cells that can be achieved. The culture of motor neurons or axons and skeletal musclecells obtained from the embryonic spinal cord explants, is easy to form functional synapticwhich can achieve a high degree of structural and functional differentiation. By now,researches about separated cells culture for synapse formation in vitro have achieved somekey results.In the research past, Chen et introduced a NG108-15/C2C12co-culture model inorder to determine the aggregation of acetylcholine receptor (AchR) clusters on the surfaceof the myotubes,as the earliest markers of the neuromuscular junction formation.AlthoughNG108-15cells can grow axons and extends to the nearby myotubes, because NG108-15cells are fused cells of the mouse neuroblastoma cell tumor cells and rat glioma cells fusedcells,the features and functions of the state are far from being able to completely replace neurons in primary cells, therefore, in co-cultured for3days,NG108-15cells graduallyapoptosis and the AchR aggregation was significantly reduced.Other researches alsoproved that rat neurons-muscle co-culture can form a large number of early NMJ,and candetect AchR aggregation on the postsynaptic membrane induced by axons，but due to thedifficulty in achieving the primary skeletal muscle satellite muscle cells,the complicationof the purification process,the high culture conditions and after spreading to ninegenerations the cell come to present senescence characteristics, can t unlimited-generationsubculture.Therefore, in this experiment, we try to improve the coculture appropriately based onthe methods introduced above, the first time to using primary fetal SD rat anterior hornspinal cord motor neurons and C2C12cells for coculture,in order to establish a a stableNMJ model in vitro.The operation in this coculture system is more simple,the repeatabilityis better, the state of the cells is well, the survival time is longer and it is easier toanalyze,so that this new system can lay the foundation for further study the functionalstatus and renewabl of the neuromuscular joints in vitro and in vivo.Part1. Isolation and culture primary embryonic rat spinal cord anteriorhorn motor neuronsObjective: To extract and culture the primary neurons skillfully, and do purificationand identification on it, in order to obtain the embryonic rat spinal cord anterior horn motorneurons that required for the formation of neuromuscular junction in vitro.Methods：Use14d~16d SD rats at pregnancy,remove the embryonic rat ventral halfspinal cord tissue,dispersed into single cells by trypsin digestion and mechanical crushingmethod of treatment, purified the motor nerurons by the differential adhesion wall,placedin a serum-free limiting medium culture,make sure the cells to survive, grow and multiply,observed the neuronal morphology, changes in the structure under an inverted microscope,and adopt immunofluorescence to identify the motor neuron by detecting the the specificmarker of the spinal motor neuron Choline acetyltransferase polyclonal antibody(ChAT).Result：It is easy to harvest cells from pregnant14d~16d rat, and the number ofmotor neurons obtained is big, the state of the cells is well and thriving, jointed withNeurobasal+2%B27medium,the cultured spinal cord anterior horn motor neurons arewith high purity,about85%,able to survive under vitro culture conditions around7~10d,and the ChAT reaction results are positive.Conclusion：Successfully isolated,cultured and got high purity anterior horn motor neurons by using pregnant14d~16d rat s ventral half of the spinal cord jointed withserum-free specific medium, ChAT can specific marker the spinal cord anterior horn motorneurons.In the first five to seven days of the culture,the number of the NMs is steady with nosignificant reduction,provide a reliable source of motor neuron for the topic to beexecuted,also for the subsequent co-culture system establishment to create the conditionsin time.Part2. Establish a myoblasts C2C12into myogenic differentiation modein vitroObjective: complete the methods for myoblasts C2C12cultured in vitro,observe theof biological characteristics of cells proliferation, differentiation and fusion of myoblasts.Methods：culture of myoblasts C2C12in vitro,in the proliferation culture medium(GM) containing10%fetal calf serum.When the cells were grown to a density of60%to70%,converted into the differentiation culture medium containing2%horse serum (DM) toinduce differentiation. Change of the morphological characteristics of the various stages ofskeletal muscle cells was observed under an inverted microscope,then take thephotographes.Using anti-myosin heavy chain antibody MHC to specific marker themyotubes by immunofluores-icence.Result：When confluented to60%to70%,cultured in vitro,C2C12cells started tomer-ge to form myotubes and the express MHC proteins.About5d to formate maturemyotubes. MHC specific reaction positive.Conclusion：Myoblasts C2C12can be stably passaged when cultured in vitro,and canbe induced to differentiate into mature myotubes by differentiation culture mediumcontaining2%horse serum (DM) with no special induction method, Providing a stablesource of skeletal muscle myotubes for establishing a co-culture system.Part3. Establishment of a motor neurons-skeletal muscle cellsco-culture systemObjective: Obtain the requirement for motor neuron-skeletal muscle cellco-culture,establish a motor neuron-skeletal muscle cells co-culture system, form NMJ invitro.Methods: The C2C12myoblasts were cultured in vitro,differentiation medium (DM)induced differentiation, they begin to differentiate fuse to form myotubes when cultureabout3d.Take the pregnant14d~16d SD rat, extract the embryonic rats al cord anterior horn motor neurons, planting to the myotubes after differentiating5days,co-culture in the differentiation culture medium containing2%horse serum(DM).Neuronal morphology and the projection change in the length of each stage,myotubes morphological characteristics change and shrinkage characteristics and theformation of the neuromuscular junction are observed under an invertedmicroscope.Observate the position and the distribution of AchRs on the postsynapticmembrane by incubating alpha-bun-BTX) together with the Living cells,at thesame time record the muscle contraction phenomenon in the co-culture by screen recordingtechnology,which is another evidence to prove NMJ formation after co-cultur.Result: Replaced by co-culture medium,both SKMs and the MNs could survive andfurther differentiation and maturation.In the co-cultured cells, MN is easy to identify inmorphological.Approximately4h after co-culture, spinal cord motor neurons begin toadherent under the inverted microscope,showing as a single round or oval,after12h, themajority of the cells were adherent,after24h, NMs gradually formed protrusions,myotubesfurther integration thickening and to form a network-like structure with each other,at thefirst3d~5d, neurons grow vigorously,with full cell body,significant halo,moreprojections.A part of the the NMs start to form connections with myotubes; about oneweek, NMs axons extending to the the myotube membrane surface or surroundingmyotubes are visible,myotubes row in the same direction, widely rhythmic constriction canbe received;about10d after co-culture,motor neurons begin to apoptosis,the myotube cellsgradually shrink.Conclusion：In vitro culture conditions,motor neurons and skeletal muscle cells canbe co-existence, growth and further developmen,further establish synapticconnections,trigger a series of neuromuscular junction signal transduction,thus causerhythmic contractions of the myotubes.