The Relationship Of The Plasticity Of Synaptic-like Structure To CaMKⅡ And To SynCAM1 In The Process Of Adipose-Derived Stromal Cells Differentiation Into Neuron-like Cells

Objective To analyze the feature of processes and synapses,and the function of synaptic plasticity during the adult adipose-derived stromal cells(ADSC)induced to differentiate into neurons,so as to provide a theoretical basis for obtaining the optimum ADSC-derived neurons and optimizing induction scheme.Methods 1 ADSC extracted from subcutaneous adipose tissue were induced to differentiate into neurons using β-mercaptoethanol(BME).2 We observed the processes morphology of the cells non-induced,pre-induced for 24 h and induced for 1,3,5 and 8h by inverted phase contrast microscopy.3 Immunocytochemistry detected the expression of neuron-specific markers--neuron specific enolase(NSE)and Microtubule associated protein 2(MAP-2)in each group cells.4 Ultrastructural features of the cells induced for 5h were observed using transmission electron microscopy(TEM).5 Immunofluorescence double staining and western blotting determined the location and expression level of NSE,synaptophysin(SYN),postsynaptic density 95(PSD-95),calcium/calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ),phospho-Ca MKⅡ(p Ca MKⅡ)and Synaptic cell adhesion molecules 1(Syn CAM1).Results The induced cells connected each other forming network structure,with cytoplasm contracting,refractivity enhanced and the number of protrusions increasing.The number of total processes and of long processes were largest at 5h,while both significantly decreased at 8h(P=0.000,0.005,respectively).Meanwhile,the neuronal differentiation rate peaked at 5h by(87.33±1.53)%,but there was no significant difference with 8h(P=0.652).Immunofluorescence and western blotting showed that NSE,SYN and PSD-95 did not been expressed in ADSC,but in induced cells and peaked at 5h.NSE had no significant difference between 5h and 8h(P>0.05),while SYN and PSD-95 in 8h were significantly lower than 5h(P<0.05).Similarly,Ca MKⅡ,p Ca MKⅡ and Syn CAM1 also reached the peak at 5h,and deduced at 8h(P<0.05).Under TEM,the cells induced for 5h were observed not only having a lot of Nissl bodies and a mass of synaptic vesicle-like structures in the cytoplasm near the cell membrane,but also presenting high density on the side of the membrane of two junction cells.Conclusion It was at 5h that the induced cells not only had typical neuronal characteristics in morphology and structure,but also had the largest number of protrusions,synaptic ultrastructure and strong synaptic plasticity.If the cells were further induced,the number of protrusions and synaptic plasticity would significantly decrease.Besides,Ca MKⅡ and Syn CAM1 played important roles in synaptic plasticity during the process of ADSC differentiation into neurons.