| Background:Inhibitory microenvironment in spinal cord injury region is an important factor hindering axon regeneration and rehabilitation.Although neural stem cell transplantation has achieved good results,the problems of ethics,cell source,immune rejection and tumorigenicity of exogenous cells restrict clinical transformation.How to regulate endogenous neural stem cells has become the focus of scholars.Studies have confirmed that microRNAs are widely expressed in the central nervous system and play a role in regulating neural stem cells.Researchers believe that synthetic microRNAs can be introduced into cells to play a regulatory role.However,synthetic microRNAs are readily enzymatic hydrolysis in the spinal cord injury area,and negatively charged microRNAs are difficult to transport across the membrane.Therefore,it is necessary to construct a vector to avoid the enzymatic hydrolysis of microRNAs and transports them across the membrane to regulate endogenous neural stem cells.Objective:Based on the previous studies,we intend to prepare nano-carriers of miR-29 and implant self-assembled polypeptide scaffolds into the spinal cord injury area of rats to explore the role and mechanism of these nano-carriers in regulating endogenous neural stem cells,so as to provide new ideas and research basis for the development and clinical transformation of self-assembled nano-polypeptide microRNA sustained-release system.Method:1.Establish rat spinal cord injury model;BBB score and histopathology at each stage;the proliferation and differentiation of neural stem cells were detected by immunofluorescence;the expression of miR-29,Nestin gene and protein were detected by RT-PCR and WB.2.The surface of nano gold particles was modified by oxidation-reduction method to prepare PEG-SH modified GNPs.Ultraviolet absorption spectrophotometer was used to detect absorption peak;transmission electron microscopy detected particle size distribution;Zeta potential detected surface potential;synthetic microRNA and PEG-SH GNPs/miR-29 nanoparticles were detected by SDS-PAGE gel electrophoresis at different time intervals;CCK-8 method was used to detect the activity and proliferation of neural stem cells among synthetic miR-29,PEG-SH GNPs,PEG-SH GNPs/miR-29 nanoparticles3.The peptide was synthesized by solid phase method,the PH of the peptide was adjusted to the critical point of self-assembly to form gel,and detected by scanning electron microscope and transmission electron microscope.Neural stem cells were inoculated on scaffolds and pre-coated polylysine slides.DMEM/F12 and 1%fetal bovine serum were dripped into the cell culture plate and cultured.Differentiation was detected by immunofluorescence.PEG-SH GNPs/miR-29 composite nanoparticles were constructed and their suspensions were slowly added to the scaffolds.Cy5-labeled PEG-SH GNPs/miR-29 nanoparticles were gradually dripped onto the scaffolds to study the distribution of microRNAs on the scaffolds.The freeze-dried scaffolds were observed under scanning electron microscope.Quantitative detection of microRNAs was performed at 0,2,4,6,8,10,12 hours to study the release of microRNAs on scaffolds.Immunofluorescence detection was performed after dripping culture medium and serum.Axon regeneration was measured by Image J software.Immunofluorescence was used to detect axon regeneration after transplantation of self-assembled nanopeptide miR-29 sustained-release system into spinal cord injury area.The changes of BBB score of motor function in rats after transplantation of self-assembled nano-peptide miR-29 sustained-release system were recorded at given time interval of spinal cord injury.Result:1.The expression of miR-29 decreased significantly at the early stage of injury,then increased gradually,reaching its peak at 21 days;the expression of Nestin was almost the same as that of miR-29;neurons increased at the late stage of injxury and motor function improved in rats.2.Gold nanoparticles modified by PEG-SH were successfully prepared.Under transmission electron microscopy,they were spherical with uniform particle size distribution and kept in a diameter of 20-25 nm.With the increase of PEG concentration,Zeta potential gradually increased to a peak of 22.5+5.2 mv?and PEG could effectively adsorb microRNA with negative charge.In 0-6 hours,synthetic miR-29 had clear expression bands,and in 12-24 hours,miR-29 expressed bands quickly disappeared.In PEG-SH GNPs/miR-29 group,the expression of microRNA bands was stable.CCK-8 showed no sigrificant difference in the proliferation of NSCs in each group.3.self assembled peptide gel has good viscoelasticity and good porosity can be seen under the electron microscope.In vitro experiment can induce the proliferation and differentiation of neural stem cells.Under the microscope,miR is evenly distributed on the surface and inside of the scaffold,and miR shows a sustained release effect on the scaffold.Endogenous axon regeneration can be found after transplantation of self-assembled nanopeptide miR-29 sustained-release system.The BBB score did not change significantly on the first day after the implantation of the material into the spinal cord injury area.On the third day,the BBB score increased rapidly,peaked on the seventh day and remained stable until the twenty-eighth day.Conclusion:1.The expression of miR-29 is related to the proliferation of endogenous neural stem cells at different stages of spinal cord injury in rats;the increase of miR-29 expression can improve motor function.2.PEG-SH modified gold nanoparticles have good particle size and surface potential,which can effectively adsorb and prevent the degradation of microRNAs without affecting the proliferation of neural stem cells.3.miR-29 was uniformly distributed on the scaffold and released slowly.Both in vitro and in vivo experiments confirmed that it was promoted axon regeneration and improved hind limb motor function in rats with spinal cord injury. |
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