The 2D And 3D Neural Transdifferentiation Mediated By Carbon Dots

Due to the safety problem of viral vectors,the research of non-viral nano-gene vectors has attracted much attention.In recent years,many studies have reported the use of cationized polysaccharides as gene carriers deliverying specific genes for cell reprogramming.Natural polysaccharides enjoy good safety and low cytotoxicity.Therefore,the polysaccharide-derived cationized carbon dots can not only transfer gene safely,but also visualize the gene transfer process by utilizing the fluorescence properties of carbon dots.The cationized carbon dots are expected to be developed as a novel non-viral gene vector with both gene delivery and bioimaging functions.However,the studies on gene delivery and cell differentiation mediated by carbon dots are not in-depth enough.Therefore,in this study,fluorescent carbon dots were synthesized using a one-pot hydrothermal reaction,with the natural porphyra polysaccharide serving as a single carbon source and ethylenediamine（Ed）acting as the surface passivation agent.After investigating the properties of carbon dots,we used the carbon dots as non-viral gene vectors combining and delivering the plasmid Ascl1,Brn2 and Sox2 to induce ectodermal mesenchymal stem cells（EMSCs）into neuron-like cells in vitro so we can use the neuron-like cells as seed cells for the treatment of neuronal injury caused by injury or diseases.In addition,cells mostly culture in two-dimensional environment in vitro and it is different from the cells culturing in vivo.Nerve damage caused by external force,such as spinal cord injury,usually need the biocompatible tissue engineering scaffolds to support cell adhesion,proliferation and migration so the scaffolds can carry cells and transplant into the injury for treatment.The suitable tissue engineer scaffolds can anti-apoptosis,anti-inflammation and promote nerve regeneration.In this study,hydrogels were prepared by sodium alginate and gelatin,and we screened a suitable one from the 45 groups hydrogels.This study provided a preliminary basis for the transplantation of the optimized hydrogel loaded the induced nerve cells into the semi-transection of the spinal cord of rats to repair the spinal cord injury.Chapter one:ReviewWe review the preparation,properties and current applications of carbon dots,expecially the excellent optical properties and bioimaging,so as to provide theoretical references for using carbon dot as a multifunctional nano-gene vector.In addition,the methods of obtaining induced-nerve cells in vitro,including directed differentiation of pluripotent stem cells and somatic cells reprogramming,are briefly introduced to provide basis for using carbon dots to carry specific plasmids and transfect EMSCs.Then,we introduce the currently biological scaffolds applied to treat spinal cord injury,and provide references for the selections.Lastly,the theoretical basis and ideas of the topic selection are proposed to lay the foundation for the work in this paper.Chapter two:Synthesis and characterization of carbon dotsIn this study,cationic carbon dots（CDs）were synthesized using a one-pot hydrothermal carbonization reaction,with the naturally-occurring porphyra polysaccharide serving as a single carbon source and ethylenediamine（Ed）acting as the surface passivation agent.We investigated the fluorescence properties of CDs by measuring uv-vis absorption spectra,fluorescence emission and excitation spectra,and fluorescence quantum yield.In addition,we investigated the surface modification and composition of CDs by infrared spectra and determination of nitrogen.The CDs enjoyed excitation-dependent fluorescence,trichromatic fluorescences（display red,green and blue fluorescence under the different excitation wavelengths）,positive surface charge,high quantum yield（56.3%）and excellent optical properties.Chapter three:Study on the properties of carbon dots as a gene vectorIn this chapter,we mainly focused on the gene delivery capacity of CDs.The ability of cationic CDs combining plasmid DNA was examined by agarose gel electrophoresis,and the result demonstrated that the plasmid DNA was completely retained in the original well and combined with CDs efficiently when CDs/plasmid DNA weight ratio was 10:1.We measured the particle size,Zeta potential and scanning electron microscopy of CDs/pDNA nanoparticles.The particle size of CDs was around 4 nm and the CDs/pDNA nanoparticles were around 14 nm.Both CDs and CDs/pDNA nanoparticles were positively charged and uniformly dispersed.In addition,the cytotoxicities and cellular uptake pathways of CDs/pDNA nanoparticles were examined.Compared with cationic transfection reagents,CDs/pDNA nanoparticles transported into cells by clathrin and caveolae-dependent endocytosis with lower cytotoxicity.Chapter four:Study on the neuronal differentiation of ectodermal mesenchymal stem cells via CDs/pDNA nanoparticlesIn this chapter,the CDs was used as a gene vector,and the seven groups of plasmids consisting of Ascl1,Brn2 and Sox2 were combined to form CDs/pDNA nanoparticles which were used to transfect the ectodermal mesenchymal stem cells（EMSCs）.The optimal transfection combination was determined by observing the cell morphology,ELISA and Western blot.Lastly,the neuron-like cells were identified by immunofluorescence,immunohistochemistry and RT-PCR.Ascl1 and Brn2 were selected as the optimal combination to induce EMSCs to differentiate into neuro-like cells which expressed neuron markers Tuj1,Map2,Tau,Gap43 and NF200.Compared with the commercial transfection reagents（including PEI and Lipofectamine2000）,CDs exhibited significantly higher transfection efficiency.Chapter five:Preliminary study on three-dimensional culture system In this chapter,sodium alginate and gelatin were used as raw materials,andCaCl₂ was used as the coagulant.The properties and biocompatibility of the 45 groups of hydrogels were initially evaluated by measuring gel-time and cytotoxicity.Next,the water absorption,swelling rate and degradation rate of the hydrogel were measured,and the physical properties of the hydrogel were examined to further select the optimum hydrogel formulation.Finally,the transfected EMSCs were mixed with the composite hydrogel to form a cell-loaded hydrogel.The survival and growth of the cells on the hydrogel at different culture time were observed.In addition,the neural differentiation efficiency between 2D and 3D cell culturing was compared so as to decide whether the hydrogels were suitable for cell growth and provide a basis for transplantation of cell-carried hydrogels in vivo.