Synaptic Release Of Adipose Stromal Cell-derived Neurons:A Molecular Structure Study

Objectives To explore whether β-mercaptoethanol-induced adipose-derived stromal cells neurons have the molecular basis and structural basis related to synaptic release function of normal mature neurons.Methods 1 ADSCs were extracted in vitro and cultured and passaged.2 The thirdgeneration ADSCs were induced to differentiate into neurons by βME.3 Identification of ADSC-derived neurons: Immunocytochemistry and Western blotting were used to detect the expression of NSE and MAP2 of the cells in the ADSCs group and in the induced groups for 1h,3h,5h and 8h.4 Immunocytochemistry,Immunofluorescence and Western blotting were used to detect the expression of Synapsin I,VAMP2,syntaxin 1A,SNAP-25 and Synaptotagmin 1 of the cells in each group.5 Ultrastructural features of ADSCderived neurons at 5h after differentiation were observed by transmission electron microscopy(TEM).6 Statistical Analysis: All experimental data were recorded by Excel 2007 and statistical analysis was performed by SPSS software(version 17.0).A single factor analysis of variance was used for F test and P <0.05 was considered statistically significant.Results 1 The initially extracted ADSCs adhered within 24 hours.The cells were round or irregular.With the extension of cultivation time,ADSCs gradually showed a shuttle form and whorled in arrangement.ADSCs were completely purified and vigorously grown to induce differentiation when cultured to the third generation.2 After induction for 1h,the cytoplasm retracted and the protuberances lengthened.After a 3h induction,some ADSC-derived neurons showed a multipolar neuron-like cell morphology.After a 5h induction,the dendritic-like branches of the ends of some long protuberances of ADSC-derived neurons were interconnected to form a net.ADSC-derived neurons showed a typical neuron-like morphology,which dominated by bipolar neuron like cells,and the number of multipolar neuron like cells increased further.After a 8h induction,the morphology of ADSC-derived neurons degenerated.3 ADSCs did not express NSE and MAP2,while they were expressed in each induction group.The positive rates of NSE and MAP2 reached the peak at 5h but there was no significant difference with 8h(P<0.05,respectively).4 Immunocytochemistry and Western blotting showed that ADSCs did not express Synapsin I,VAMP2,Syntaxin 1A,SNAP-25 and Synaptotagmin 1,while they were all expressed in each induction group.The positive rates and expression levels of Synapsin I and Syntaxin 1A reached the peak at 8h,while VAMP2,SNAP-25 and Synaptotagmin 1 reached the peak at 5h and there were significant differences in each group(P<0.05,respectively).The positive rates and expression levels of VAMP2 and SNAP-25 were not statistically different between the 8h and 5h groups(P>0.05,respectively);The positive rate and expression level of Synaptotagmin 1 decreased in the 8h group compared with the 5h group(P<0.05).5 Immunofluorescence showed that ADSCs did not exist NSE/Synapsin I,NSE/VAMP2,NSE/Syntaxin 1A,NSE/SNAP-25 and NSE/Synaptotagmin 1 co-positive cells,while they existed in each induction group.The co-positive expression ratios of NSE/Synapsin I and NSE/Syntaxin 1A reached the peak at 8h,while NSE/VAMP2,NSE/SNAP-25 and NSE/Synaptotagmin 1 reached the peak at 5h and there were significant differences in each group(P<0.05,respectively).6 The Transmission electron microscopy revealed that the ADSC-derived neurons induced for 5h had a lot of microvilli and projections on the surface,and had rich organelles,as well as Nissl body could be seen.In the cytoplasm of ADSC-derived neurons,a large number of round vesicles with different density and volume were observed,especially in the vicinity of the cell membrane,and the vesicle membranes of some vesicles are similar to the fused part of the cell membrane in a released state.Conclusion ADSC-derived neurons have typical neuronal synaptic vesicle-like structures and synaptic vesicle release function related Synapsin I,VAMP2,Syntaxin 1A,SNAP-25 and Synaptotagmin 1,ADSC-derived neurons have synaptic vesicle mobilization,activation and membrane fusion functions in normal neurons.