| The developmental process of hippocampal neurons is mainly including the growth of the growth cone and neurites,the formation of dendritic spines,and the pruning of functional synapses,et al,which is very important to the ability of learning and memory.Many studies have provided evidences for that multiple factors are involved in the development of hippocampal neurons.Recent studies have shown that graphene can promote cell growth due to its good electrical conductivity and large specific surface area.Graphene-noble metal nanocomposites not only maintains the biosafety,excellent electrical,optical,mechanical properties and great specific surface area of grapheme,but also are imparted many properties such as catalytic properties and enhanced photothermal effects.However,there are still no reports on the application of reduced grapheme oxide graphene-noble metal nanocomposites in the field of neuroscience.Therefore,this study selected reduced grapheme oxide@palladium nanocomposites(rGO@Pd)which was given the catalytic hydrogenation ability of palladium as a growth substrate for hippocampal neurons,to explore the effects of rGO@Pd nanocomposites on the development of hippocampal neurons.We observed the growth cones and neuronal neurite,and further explore the effect and its relevant mechanisms of reduced grapheme oxide graphene-noble metal nanocomposites on development of hippocampal neurons,in order to provide novel strategies for promoting the growth of neurons or treating diseases related to neuronal development as well as potential cues for clinical work.My work is as followed:Objective: To observe the effects of rGO and rGO@Pd nanocomposites on hippocampal neurons and explore the underlying mechanisms.Methods:(1)Primary cultured hippocampal neurons were obtained from E18 of C57BL/6 mice.The experiment was divided into control group,the rGO group at different concentrations(0.01,0.02,0.05 and 0.1 mg/mL),and the rGO@Pd group at different concentrations(0.01,0.02,0.05 and 0.1 mg/mL).(2)The microtubule-associated protein-2(MAP-2)and glial fibrillary acidic protein(GFAP)antibodies were used for immunofluorescence to identify the cells.(3)The dendritic spines were observed by liposome transfection of GFP(Green fluorescent protein)and the synapses were immunostained with antibodies against the presynaptic marker synaptophysin and the postsynaptic marker PSD-95.(4)rGO@Pd nanocomposites were obtained by ultrasonic treatment of rGO@Pd nanocomposites and characterized by Transmission Electron Microscopy(TEM),Scanning Electron Microscopy(SEM),Zeta potential map and Fourier Transform Infrared Spectroscopy(FTIR);(5)The neuronal viability was detected by CCK-8 assay and Calcein-AM/PI live cell/dead cell double staining kit;(6)Hippocampal neurons were stained with antibodies ?-tubulin and TRITC-Phalloidin 24 h after seeding onto the substrates,to detect the growth of growth cones;(7)Cells were immunostained with antibodies against ?-tubulin,to visualize the morphology and dendritic complexity of hippocampal neurons.Results:(1)On the 4th day of neuron cultivation,the neurons showed plumpness with clear background and few pyknosis dead cells.(2)The positive rate of immunofluorescence staining of specific marker for neuron,MAP-2,was above 90%;(3)The morphology of dendritic spines were observed at different developmental stages by transfection of GFP with liposome on the 12 th day of neuron cultivation;Highly differentiated axons,dendrites,spines and functional synaptic connections could be observed by synaptic staining of hippocampal neurons cultured for 21 days.(4)The characterization of rGO@Pd nanocomposites showed that rGO@Pd nanocomposites used in this experiment were uniform and microporous structures,and the palladium particles have dispersed to the surface of rGO uniformly.The functional groups of rGO@Pd nanocomposites have almost disappeared and the oxygen-containing groups were completely reduced,indicating that the material is rGO@Pd nanocomposite.In addition,it had good stability and dispersibility in water.(5)CCK-8 assay and Calcein-AM/PI test showed that rGO group at 0.02 or 0.05 mg/mL,rGO@Pd group at 0.02 or 0.05 mg/mL,increased neuronal viability,compared with the control group(P<0.05);cell viability of rGO@Pd group at 0.05 mg/mL was higher than the rGO groups at 0.01 mg/mL(P<0.05);(6)Immunofluorescence staining of phalloidin and ?-Tubulin showed that the area of neuronal growth cone with treatment of rGO@Pd nanocomposites were significantly increased compared with the rGO group(P< 0.05)and PDL control group(P< 0.01);(7)Immunofluorescence staining of neuronal neurite showed that the length of it in rGO@Pd group with 0.05 mg/mL were significantly higher than those in both control group(P< 0.01)and rGO group(P< 0.05).Conclusion:(1)We have built a stable and efficient method culturing mouse hippocampal neurons with high purity,low morality,high yield and low background.(2)Using rGO@Pd nanocomposites(0.05 mg/mL)as a growth substrate of mouse hippocampal neurons can greatly improve neuronal viability,promote the growth of growth cones and enhance the dendritic length. |
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