| Objectives To investigate whether adipose-derived stromal cells(ADSCs)derived neurons have neuronal synaptic endocytosis.Methods 1 Waste adipose tissues were obtained via an needle-suction procedure from adult subcutaneous adipose tissue,and ADSCs were extracted and cultured.2 The third generation of ADSCs were selected,and ?-mercaptoethanol(?-ME)was used to induce differentiation into neurons.The cells were divided into ADSCs group,1h,3h,5h,and 8h groups.3Detection of specific markers of ADSCs-derived neurons: immunocytochemistry and Western-blotting detection of neuron-specific enolase(NSE)and microtubuleassociated protein(MAP-2).4 Detection of endocytotic markers in ADSCs-derived neurons: immunocytochemistry,immunofluorescence and Western-blotting for localization and quantitative detection of neuron endocytosis core protein adaptor protein(AP-2),Clathrin,Endophilin,Dynamin and heat shock cognate protein(Hsc70).5 Three-dimensional morphological changes of ADSCs during differentiation into neurons were observed by scanning electron microscopy.6 Ultrastructural characteristics of synapses during ADSCs differentiation into neurons were observed by transmission electron microscopy.7 Synaptic endocytosis test of ADSCs-derived neurons: FM1-43 neuron synaptic endocytosis function test and cadmium selenide quantum dots(Cd Se QDs)neuron synapse endocytosis function test were used to detect whether the differentiated cells had endocytosis.8 Statistical analysis: data obtained were stored in Excel 2007.SPSS 17 was used for statistical analysis.The data were expressed as meansąstandard deviations.One-Way ANOVA was used for multiple group comparisons and LSD test or Tamhane's T2 test were performed for post hoc paired comparisons.Statistically significance was set at P< 0.05.Results 1 ADSCs adhered to the wall within 24 hours after extraction and cultivation,which could be irregular,round,and polygonal.With the increase of culture time,ADSCs gradually changed into a long spindle and gathered in a vortex.The fully purified ADSCs passaged to the third generation mainly showed regular long spindle-shaped dense growth and showed a sheep-like distribution trend.2 Results of ADSC-derived neuronal specific markers: NSE and MAP-2 were not detected in the uninduced ADSCs group,but only in the differentiated groups.The result showed that NSE and MAP-2 in each group of cells had the strongest expression levels during the 5h of induction(all P<0.05).3 Detection results of synaptic markers of ADSCs-derived neurons:there were no AP-2,Clathrin,Endophilin,Dynamin,and Hsc70 protein expressions in uninduced ADSCs.As the time of induced differentiation was prolonged,the positive expression levels of AP-2,Clathrin,Dynamin,and Hsc70 all peaked at 5h(P<0.05),but the expression levels of Endophilin peaked at 5h,but there was no significant difference with the 8h group(P>0.05).4 Scanning electron microscopy showed the 3rd-generation ADSCs of fusiform growth,with the round cell body and uniform reflection,and no protrusions at both ends of the cells.Induction at 1h showed the fusiform cell body shrinkage,being thinner and longer,and bipolar protrusions of varying lengths.Induction at 3h caused further cell body shrinkage,a long protrusion from the cell body,with a slightly enlarged endfoot,and several shorter protrusions with small dendritic branches.At 5h,the number of protrusions from the cell body increased more significantly,and a more slender protrusion with swelling endfoot and numerous microvilli of varying thickness and length were observed.At 8h,the neurite outgrowth branches were reduced,broken and the cell morphology was degraded.5 Transmission electron microscopy showed that ADSCs was smooth and intact,the nucleus was regular and several organelles were scattered in the cytoplasm.Induction at 1h induced irregular small protuberances began to appear on the cell surface,and a small number of organelles such as lysosomes appeared in the cytoplasm.Induction at 3h indicated that the irregular small protuberances on the cell surface were gradually increased and prolonged,a significantly increased number of vesicles were seen at the end of the process,with the presynaptic membrane-like structures contained many mitochondria and vesicles and the postsynaptic membrane-like structures showed similar or higher electronic density than elsewhere.At 5h saw that a large number of microvilli and relatively long protuberances appeared on the cell surface.Meanwhile,a large number of vesicles with various shapes were seen at the protuberances.The cytoplasm was rich in organelles and was rich in the specific organelle structure of Nissl bodies.At 8h,the number of microvilli and protuberances on the cell surface was significantly reduced,organelles were swollen,and secondary lysosomes were abundant.6 Test results of synaptic endocytosis of ADSCs-derived neurons: FM1-43 neuron synaptic endocytosis test results showed that FM1-43 positively expressed extracellular solution and cell membrane in ADSCs group.After induction,the vesicles in the cells of each group showed a circular membrane-like structure around the FM1-43 positive expression and intermediate negative expression,and the expression levels of FM1-43 peaked at 5h.Cd Se QDs neuron synaptic endocytosis test results showed that Cd Se QDs were positively expressed in the cytoplasm in the ADSCs group.Cd Se QDs positive granule like structures were seen at the protrusions of the cells in the induced differentiation groups,and the expression levels of Cd Se QDs peaked at 5h.Conclusion ADSCs-derived neurons have thephysiological characteristics of normal neurons,and have vesicle-like ultrastructure characteristics at the end of the protuberant and the core proteins AP-2,Clathrin,Endophilin,Dynamin and Hsc70 required for the function of synaptic endosytosis,and FM1-43 and QDs neuronal functional tests have verified that ADSC-derived neurons have the function of normal synaptic endosytosis.Figure18;Table12;Reference 89... |
PDF Full Text Request