The Gene Delivery System Mediated By Carbon Quqntum Dots For Stomic Cells Differentiation Into Neural Cells

The direct reprogramming can supply safety cell source for alternative therapy,which is considered to be one of the best efficiency strategies of CNS impairment,by direct conversion of somatic cell to induced-neural stem cells(iNSCs)or induced-neuronal(iNs).However,viral vectors may increase the risk for undesired mutagenesis and tumor formation.Carbon dots(CDs)is a kind of fluorescent carbon nanoparticles with little size(<10nm),it is widely applied into medical and biological field because of its excellent fluorescent properties,good biocompatibility and low cytotoxicity,such as fluorescent probes,biological imaging biochemical detection,drug delivery and so on,but seldom been employed as gene vector.Here,we describe one-step hydrothermal carbonization route with alginate as raw material to prepare carbon quantum dots(CQDs),which is involved into direct conversion of somatic cell to induced nerve cells as gene delivery.Chapter one: ReviewsThe carbon quantum dots of different synthesis methods,properties and their application in various fields are discussed.We focus on the advantages and disadvantages of different methods to prepare carbon quantum dots,as well as application prospects of different areas,aimed to provide a reference point for further research as gene vector.Meanwhile,we review the recent advances in generation of iNSC and iNs in vivo,which includes the features of different cell resource and different reprogramming strategies,and their development trend and challenges in the future.Chapter two: Synthesis and characterization of carbon DotsWe prepared a kind of carbon quantum dots derived from alginate by one-step hydrothermal method,and its optical properties were observed with various characterization technology.It was found that the size of CDs is much smaller than 10 nm,like spherical particles.The CDs exhibited strong and stable fluorescence with water-dispersible and positively-charged properties.Interestingly,excitation-dependence was demonstrated via scanning its fluorescence emission spectrum.What's more,CDs prepared here can show three kinds of fluorescence colors,blue,green and red,respectively.Chapter three: Preliminary study on carbon dots as gene deliveryThe study on carbon dots as gene delivery were carried out,to verify the gene delivery capacity and transfer mechanism.The results of MTT and agarose gel electrophoresis demonstrated that CDs had negligible cytotoxicity and could serve as an excellent DNA condensation when the mass ratio between CDs and DNA were 40:1.Based above,we combined CDs and pDNA to form nanoparticle and then treated 3T6 cell in vivo,which found that CDs exhibited higher transfection efficiency compared with PEI and Lipo2000,and DNA could appear in cell nucleus 4 hours later with CDs keeping out of nucleus.By tracking of the cellular-uptake found that both caveolae-and clathrin-mediated endocytosis pathways were involved in the internalization process of CDs/pDNA complexes.Chapter four: Study on cell reprograming mediated by carbon dotsIn order to verify the potential of carbon dots as non-viral gene vector in cell reprogramming,CDs was combined with five different transfection factor groups respectively to direct reprogram mouse embryonic fibroblasts(MEF)to induced neuronal stem cells in vivo,then the iNSCs were directional differentiate to induced nurons.The results of MEF converted to iNSCs show that iNSCs not only like NSC in morphology,but also expressed the specific markers of NSC,nestin and pax6.and after directional differentiation,those spheres-like cell converted to neuron-like cells,as well as expressed the specific markers of neuron,MAP2,Tau and tuj1.By comparative analysis,we found that the transfection efficiency of CDs/Ascl1-Brn2 complex nanoparticle is better than other groups,suggesting Asc1 and brn2 play important roles in the process of reprogramming,by corporation together.