Study On Directional Differentiation Of Neural Stem Cells Into Neurons And Repair Of Spinal Cord Injury In Vivo Mediated By Ultra-small Cationic Carbon Quantum Dots

Using exogenous or endogenous neural stem cell(NSCs)to repair spinal cord injury(SCI)has always been a world-class challenge,mainly due to NSCs in the complicated microenvironment of the injured site tend to differentiate into astrocytes rather than neurons.And the astrocytes,in combination with their secretions,usually form astroglial scar that constitute the physical and chemical barrier of nerve regeneration.Although there are many strategies attempted to achieve directional differentiation of neurons within the lesion center,it has not been reported that ultra-small particle size cationic carbon quantum dots(CQDs)can promote the repair of SCI by inducing NSCs to differentiate into neurons.At the same time,this is also the first time to demonstrate that cationic CQDs can influence cell differentiation fate in addition to depend on their optical properties(such as biolabeling and bioimaging)and cationic properties(such as gene therapy and drug delivery).In this study,the ultra-small particle size cationic carbon quantum dots were prepared by one-step hydrothermal synthesis using Lycium barbarum L.oligosaccharide(LBOS)as carbon source,ethylenediamine and PEI as cationic reagents.The in vitro experiments showed that the ultra-small particle size cationic CQDs could significantly inhibit the differentiation of NSCs into astrocytes and promote the extension of neurons to the surrounding area without affecting the differentiation of neurons.The in vivo experiments indicated that the ultra-small particle size cationic CQDs could significantly inhibit the differentiation of endogenous and exogenous NSCs into astrocytes in the area of complete spinal cord transection,thus inhibiting the hyperplasia of astroglial scar and promoting the reconstruction of neural circuit.Collectively,the in vitro and in vivo experiments revealed that the ultra-small particle size cationic CQDs derived from LBOS could significantly inhibit the differentiation of NSCs into astrocytes,thus directing the directional differentiation of neural stem cells into neurons.Chapter one: ReviewsThis chapter focuses on spinal cord injury,and emphasizes the pathological changes of the injured area after SCI,the construction of SCI animal model,the therapeutic strategy of SCI and the evaluation of the treatment effects.Then,the preparation methods,optical properties and application fields of the carbon quantum dots are briefly described.In this process,we found that up to now,there have been no relevant reports showing that the ultra-small particle size cationic CQDs can promote the directional differentiation of neural stem cells.Finally,the design idea and research significance of this topic are put forward,which lays the foundation for the follow-up work of the paper.Chapter two: Extraction,purification and preliminarycharacterization of the LBOSThis chapter mainly introduces the extraction-purification process and preliminary characterization results of the LBOS,which is the carbon source of the ultra-small particle size cationic CQDs.Specifically,the LBOS were extracted and purified from Lycium barbarum L.by Water-extraction and alcohol-precipitation method,Seveg method and multistage column chromatography.Then the purity,molecular weight and monosaccharide composition of the LBOS were characterized by high performance gel permeation chromatography,Coomassie brilliant blue assay,mass spectrometry and monosaccharide composition detection.Chapter three: Preparation and characterization of the ultra-small particle size cationic CQDsIn this chapter,ultra-small particle size cationic CQDs were prepared by one-step hydrothermal synthesis using Lycium barbarum oligosaccharide as carbon source,ethylenediamine and PEI as cationic reagents.Then the optical properties,charge properties and particle size of the CQDs were characterized by UV-visible absorption spectra,fluorescence excitation and emission spectra,infrared spectra,agarose gel electrophoresis and high resolution transmission electron microscopy.The results indicated that the CQDs prepared in this study are ultra-small particle size carbon quantum dots(~ 1 nm)with positive charge on the surface.The fluorescence excitation and emission spectra showed that the CQDs could emit different fluorescence colors under different wavelengths of excitation light,which means the CQDs’ s fluorescence emission wavelength was excitation wavelength dependent.Chapter four: In vitro neuronal directional differentiation ofNSCs induced by the ultra-small particle size cationic CQDsAfter the cell was fixed and cell membrane lost its selectivity,the ultra-small particle size cationic CQDs would gradually escape from the cell due to the ultra-small particle size,and the fluorescence of the cell was greatly reduced.Therefore,it is reasonable to use immunofluorescent staining to detect the cell identity.Based on above premise,this chapter taked LBOS as control,and used GFAP(astrocyte marker),Tuj1(immature neuron marker)and MAP2(mature neuron marker)as indicators to detected the effect of the ultra-small particle size cationic CQDs on the differentiation of neural stem cells in vitro by immunofluorescence labeling and Western Blot method.The final results showed that compared with the LBOS group,the CQDs prepared with LBOS as carbon source could significantly inhibit the differentiation of NSCs into astrocytes and promote the extension of neurons to the surrounding area without affecting the differentiation of neurons.Then,in order to explore the necessity of using two cationic reagents in the preparation of CQDs,PEI-CQDs and ethylenediamine-CQDs were prepared under the same operating conditions with the ultra-small particle size cationic CQDs.After the two kinds of CQDs were applied to neural stem cells,ethylenediamine-CQDs had no significant effect on the directional differentiation of NSCs,while PEI-CQDs showed obvious cytotoxicity.Therefore,this result further indicated the necessity of using two cationic reagents to modify the CQDs.Chapter five: Preliminary mechanism of neuronal directional differentiation of NSCs induced by the ultra-small particle sizecationic CQDsIn this chapter,we detected the effect of the ultra-small particle size cationic CQDs on GFAP by applying the CQDs on astrocytes,so as to indirectly reveal the potential mechanism of the CQDs inhibiting the differentiation of NSCs into astrocytes.Specifically,after the CQDs were exposed to astrocytes with high expression of GFAP for 8 days,the effects of the CQDs on the expression of GFAP and β-tubulin in astrocytes were observed by double-label immunofluorescence staining.The expression range and expression level of β-tubulin could reflect the living state of cells,and then reflect the toxic effect of carbon quantum dots on astrocytes.The results showed that with the increase of the CQDs’ s concentration,more and more astrocytes expressed low or no GFAP,but had no significant effect on the expression of β-tubulin.According to this phenomenon,we speculated that the ultra-small particle size cationic CQDs could specifically bind to negatively charged GFAP after entering astrocytes,affecting the assembly of GFAP and inhibiting the expression level of GFAP.Based on this reason,we speculated again that when the CQDs applied on NSCs,the CQDs entered in NSCs will specifically bind to the negatively charged GFAP,which expressed initially when neural stem cells tend to differentiate into astrocytes,affecting the assembly of GFAP and inhibiting the differentiation of neural stem cells into astrocytes.Chapter six: Repair of spinal cord injury mediated by the ultra-small particle size cationic CQDs in vivoIn this chapter,the ability of the ultra-small particle size cationic CQDs to direct the differentiation of NSCs in vivo was evaluated by the repair effect of the CQDs on rat model of complete transected spinal cord injury.Specifically,the fibrin scaffold embedded with the CQDs and exogenous NSCs was transplanted to complete transected spinal cord injury rats and the rats were allowed to repair for two months.the recovery of motor function and the regeneration of neurons in the injured area were identified by open field trajectory test and histological staining.The results showed that when the CQDs and exogenous neural stem cells were both used within the lesion center,the ultra-small particle size cationic CQDs could obviously induce endogenous and exogenous NSCs to differentiate into neurons and realize the interconnection of neurons at both ends of the injured spinal cord,Laying a cellular foundation for the conduction of nerve impulses.More importantly,when the hind limbs in other groups rats were still in drag movement,the rats treated with the CQDs and exogenous NSCs have been able to achieve coordinated gait walking within a short distance.