| BackgroundStrong evidence from experimental animal disease models and early clinical trials suggests that neural stem cell transplantation is a viable approach for the development of clinically applicable exogenous stem cell therapies.Based on the current research progress in the field of NSC,biology and transplantation of positive results,the contemporary view is transplanted NSC as local host "factories" to produce and secrete large amounts of immune and neurotrophic factors,including those contained in the cell's outer membrane vesicles factor,NSC graft as a natural source of biological agents,can regulate and promote the central nervous system(CNS)tissue in acute or chronic tissue injury after several key function recovery.However,it is an urgent problem for nerve tissue engineering to implant NSC into damaged organ tissues more effectively and to make its secreted factors act on the damaged microenvironment more effectively.Biomaterials,cells and stimuli are the three main components of nerve tissue engineering.Biomaterials,cells and stimuli are the three main components of nerve tissue engineering.Tissue-engineered nerve materials have become a potential alternative to autologous nerve grafts for nerve regeneration and functional recovery.Various materials to improve the features and enhancements as a starting point,biodegradable,biocompatible,conductive and immune inert support the ultimate goal is to accurately simulate the extracellular matrix in our body,and through a variety of polymer,cells and growth factors to induce a kind of biological chemistry,topography,and the combination of electrical signals,thus efficiently through regeneration of nerve engraftment and neural network.The selection of suitable scaffold materials to promote the differentiation of nerve cells and non-nerve cells and the growth of axons is the key to the overall design strategy of nerve tissue engineering.Hydrogels have been shown to be a good candidate for neurogenic cell culture and differentiation in many scaffolds.Considering the inherent resistance of the nervous system to regeneration,hydrogels have been widely used to release neurotrophic factors,antagonists of nerve growth inhibitors and other nerve growth promoters.In the group,the fibroin hydrogel obtained under the action of electrostatic force was studied in the early stage.The results of electron microscopy showed that the gel had nanoscale orifices and could support the adhesion growth of neurons.Curcumin is widely used in the prevention and treatment of various diseases.In recent years,curcumin has attracted more and more attention as a neurogenic and neuroprotective agent.Curcumin,as an inflammatory conditioning factor,has been confirmed by researchers to maintain the vitality of neural stem cells and its superiority in neuronal differentiation.Objectives The purpose of this study is divided into three aspects:1.Extract and extend the stable sources of human embryonic neural stem cells,the surveyor proliferation capacity of neural stem cells,specific marker expression and differentiation ability,the use of silk fibroin aqueous solution to test whether silk fibroin influence the vitality of neural stem cells,proliferation and differentiation ability,for the later use of silk fibroin preparation hydrogel scaffold structure foundation.2.Prepare 3d oriented hydrogels through the action of electrostatic field force on silk fibroin protein,screen the appropriate hydrogel concentration,conduct adhesion culture in combination with NSC,and detect the influence of hydrogel structure on NSC's adherence and neuronal differentiation ability,especially in the study of neuronal axon development and extension orientation.A model of hypoxic-ischemic brain injury in rats was constructed simultaneously to observe the repair effect of orientation hydrogel and control group disordered hydrogel combined with NSC transplantation.3.To explore the intervention effect of curcumin as a multi-signal activated traditional Chinese medicine monomer in inflammation regulation on the nerve tissue engineering of nsc-fibroin hydrogel,providing a new idea for the study of neuroprotective mechanism and in vitro drug screening.Materials and methods1.Mechanical digestion and extraction of human embryonic neural stem cells,serum-free suspension culture to amplify nerve spheres,CCK8 to detect the proliferation capacity of NSC,flow cytometry and immunofluorescence assay to detect the expression of Nestin,Vimentin,musashi-1,Notch,SOX1,SOX2,ssea-1,CXCR4,the dry markers of neural stem cells.After culture in differentiation medium for 14 days,the expression of specific marker protein O4 in neurons Tub3,oligodendroglial GFAP and microglia was detected by immunofluorescence after morphology,and the differentiation ability was determined.Verify the properties of neural stem cells obtained by the extraction method.The effect of SF on proliferation and death of neural stem cells was detected by CCK8 and PI staining.Immunofluorescence staining was used to detect the effect of SF on NSC dryness.Electrostatic prepared.2.0.5%,1%,2% silk fibroin aqueous solution and screening of appropriate concentration of hydrogel,the NSC differentiation in the three dimensional orientation silk fibroin gel water cultivation,and the natural formation of the disordered silk fibroin gel water as control group,the Calcein-dyeing observation of cell morphology,AM living cells to detect the stretching direction of axons,differentiated by Tub3 and GFAP after dyeing and quantify the NSC's proportion of differentiation into neurons and glial cells.Use Fluo-AM dye reagent by flow cytometry instrument to detect change of calcium ion in the two cells,confocal detection axon protein Bassoon,expressed after the differentiation of synapses in the rt-pcr detection axon related genes of GAP 43,NCAM1,PSD-95-and Syp,expressed in two kinds of cells in the analysis of the orderly hydrogel impact on axons and axonal extension.3.The rat model of ischemia and hypoxia was constructed,and three groups of injury group(HI),neural stem cell treatment group(NSC)and neural stem cell binding ordered Silk protein hydrogel group(NSC+Silk)were selected,with 10 animals in each group.The model was made 7 days after birth,10 days of treatment,28 days of behavioral experiments(cylindrical cylinder,balance beam and water maze),39 days of death,fixation,section,GFAP immunohistochemistry and Tub3,GFAP and Neu N immunofluorescence staining.The hippocampal tissue was obtained for the detection of neuro-related factors BDNF and NGF by ELISA.4.Suitable concentration of curcumin was screened for NSC.CCK8 staining was used to detect the cell viability of NSC at 1mg/ml,5mg/ml,10mg/ml,20mg/ml and different concentrations.Ed U staining was used to detect the effect of curcur with different concentrations on NSC proliferation.Annexin-v /PI staining to detect apoptosis by flow cytometry.Test Cur under different concentrations,the influence of the process of the NSC differentiation into neural cells differentiation culture of 12 days,through the analysis of the software quantity neurons and neuronal axons length per unit area,collect differentiation culture 6 days and 12 days of cell samples of GAP 43,NCAM1,PSD-95 and Syp mRNA expression,analyzes the role of curcumin in nerve to differentiation.Results1.After 7-10 days,the neural stem cells extracted from the primary generation by mechanical method can form a standard nerve ball colony.The NSC in the hippocampus has a fast proliferation rate,uniform size,regular morphology,and an exponential growth period of 5-7 days.The obtained neural stem cell specific markers Nestin,Vimentin,musashi-1,Notch,SOX1,SOX2,ssea-1,CXCR4 expressed by NSC in the hippocampus.It can differentiate into neuronal morphologic cells in a differentiated culture environment and partially express Tuj1,GFAP,neuronal and glial cell specific marker proteins.2.The two solutions of silk fibroin with concentrations of 0.01% and 0.1% did not affect the multiplication time of NSC,respectively: 31.5%,38.5%,40.6%;the effects of silk fibroin solution on the mortality of NSC cells were detected,respectively: 31.5%,38.5% and 40.6% in the control group(0.01% and 0.1%),but there was no significant difference.Six neural stem cells including Nestin,Vimentin,musashi-1,Notch,SOX1 and SOX2 were positively expressed by adding 0.01% silk fibroin solution to the medium.3.Electrostatic field force with 1% concentration of silk fibroin hydrogel orientation is best suited for the NSC suspension and differentiation,and compare the disorderly hydrogel material,after the differentiation of the surface of the nerve cells in the orderly hydrogel axon stretching direction to improve consistency,orderly silk fibroin gel water can improve neuronal differentiation ratio was 10.7±3.1,according to the ratio of disordered materials 7.3±2.5 significant enhancement,chaotic neuron axon length statistical analysis gel was 105.7±10.2 nm,orderly gel was 400.3±25.3 nm,quantify the neuron axon length and statistics.The axonal length of neurons was significantly increased,and the expression of Bassoon protein and its expression in axons was increased.The expression of gap-43,NCAM1,psd-95 and Syp genes were significantly increased after 12 days of differentiation.4.The rat model of ischemia and hypoxia was successfully established.After treatment with NSC and NSC+Silk,the brain defect area decreased and the expression of glial cells GFAP decreased in the cortical area,CA1 area,DG area and CA3 area,but there was no significant change between the NSC group and the NSC+Silk group.After treatment,the proportion of neurons increased,the proportion of GFAP decreased in the hippocampus,and the expression of Neu N in the hippocampus increased after treatment,all of which showed statistical differences.The results of the balance beam test and the cylindrical barrel test showed obvious repair effect in the treatment group,while the results of the water maze test showed that the NSC+Silk group had better repair effect.The content of BDNF and NGF in hippocampal tissue significantly increased one day after treatment,and the high expression state was maintained in the later period.Curcumin acted on neural stem cells and did not affect NSC proliferation,but curcumin greater than 5mg/ml inhibited NSC proliferation.After 1mg/ml curr treatment,the Ed U marker rate increased,but there was no statistical difference.After the treatment of 2.5mg/ml and 5mg/ml,the positive rate decreased,indicating that the proliferation rate of NSC decreased.There was no significant change between 1mg/ml Cur and Cont group.The results showed that high concentration of Cur could significantly induce apoptosis of NSC cells.Concentrations of 0.5mg/ml,1mg/ml and 2.5mg/ml did not affect the differentiation morphology of neural stem cells,but concentrations of 5mg/ml and 10mg/ml significantly inhibited the extension of adherent cells after neural stem cell differentiation.After Cur treatment,the number of neurons per unit area was significantly higher than that of the control group,and the length of axons of neurons was also significantly higher than that of the control group at 12 days of differentiation.MRNA expression levels of axon-related genes gap-43,NCAM1,psd-95 and Syp were increased at 12 days compared with the control group.Conclusion1.In this study,primary hippocampal NSCs from human embryos were successfully extracted,which can be steadily amplified and identified with specific markers.NSC can successfully differentiate into neurons and glial cells,which conforms to the basic properties and biological characteristics of NSCs.2.The biosecurity of silk fibroin aqueous solution as scaffold material was tested,and the effect on morphology,proliferation ability and dry ability of NSCs was evaluated,indicating its good biocompatibility.It can be used as a follow-up nerve tissue engineering material for further research.A 1% concentration of silk fibroin hydrogel supported the proliferation of neural stem cells.The ordered silk fibroin hydrogel obtained by electrostatic action can improve the differentiation ratio of neural stem cells to neurons.Ordered silk fibroin hydrogel can enhance the expression of GAP-43,NCAM1,psd-95 and Syp genes in neuronal axons during neuronal differentiation,and promote the development and migration of synapses.Ordered silk fibroin hydrogel combined with NSC in the treatment of cerebral ischemia and hypoxia model in rats has the repair effect on motor behavior and learning and memory.3.High concentrations(>2.5mg/ml)of curcumin led to NSC cell death,while low concentrations(<1mg/ml)promoted NSC cell proliferation.1mg/ml curcumin can promote the neuronal differentiation of human neural stem cells,improve the differentiation efficiency,improve the synaptic length of neurons after differentiation and the expression of synaptic related genes gap-43,NCAM1,psd-95 and Syp. |
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