Mechanisms Of NEP1-40 Gene Modified Neural Stem Cells For Spinal Cord Injury

Objective 1.Construction of NEP1-40 gene modified neural stem cell(NSC)vector by in vitro experiment,to investigate its own differentiation potential and its regulating effects on neuronal axon growth.2.By detecting the influence of NEP1-40 overexpression on the key downstream regulatory factors of Rho A/ROCK signal transduction pathway,we can clarify the mechanism of NEP1-40-NSCs promoting axon regeneration,and provide a theoretical basis for the treatment of spinal cord injury(SCI).Methods 1.Construction of NEP1-40 gene overexpression vector: First,design primers based on the known rat NEP1-40 gene sequence,and amplify the target gene fragments by PCR.Then,the NEP1-40 gene was linked to the linear vector pc DNA3.1(+)that linearized after enzyme digestion,and the combination was transformed into bacterial competent cells.Finally,the cloned colonies that grow out are identified by PCR method,and then perform restriction endonuclease and nucleotide sequencing on the clones with positive results.The constructed overexpression vector was transfected into neural stem cells,and the expression of NEP1-40 at the transcription,translation and extracellular levels were detected by RT-PCR,Western Blot and ELISA methods,respectively.2.Cultivation,differentiation and identification of NEP1-40-NSCs: Primary neural stem cells of fetal rat were extracted from SD rats at 20 days of gestation,and using suspension culture technology for cultivation and passage.The Nestin protein as a marker of neural stem cells was detected by immunofluorescence staining.Cultured and induced adherent differentiation of NEP1-40-NSCs using different differentiation media,then use immunofluorescence to stain and identify Tuj-1,GFAP,Oligo2 and MBP proteins,and observe the staining results under an immunofluorescence microscope.At the same time,the m RNA and protein expression levels of Tuj-1,GFAP,Oligo2 and MBP were detected by RT-PCR and Western Blot.3.NEP1-40-NSCs co-culture with neurons in rat hippocampal tissue: NEP1-40-NSCs was the experimental group,and NSCs group was the control group.Intervention with 1n M concentration of Nogo-66 in the two groups,β-tubulinⅢ immunofluorescence staining was performed after 5 days of culture,and then the area of newborn axons in the hippocampus of each group was measured.Phalloidine staining was used to make the structure of growth cones and filopodia clearer.Important axon and dendritic regeneration markers such as growth-associated protein 43(GAP-43),microtubule-associated protein 2(MAP-2),and amyloid β A4(APP)were detected by Western Blot.A variety of protein substrates that regulate growth cones downstream of the Rho A/ROCK signal transduction pathway,including LIMK1,LIMK2,Cofilin,and MLCⅡ,were tested in terms of protein level,and phosphorylation degree.Results 1.The results of nucleotide sequencing and electrophoresis after double enzyme digestion prove that we have successfully obtained the NEP1-40 gene overexpression vector.After the neural stem cells were transfected with the overexpression vector,we detected the expression of NEP1-40 m RNA and protein and its expression in the cell culture medium.The results showed that the NEP1-40-NSCs group was significantly higher than the NSCs group(P<0.05).2.The primary neural stem cells of SD rats were cultured and passaged using suspension culture technology.After 3 days,neurospheres of different sizes could be seen.When they grew to the 7th day,all neurospheres were tested positive for Nestin staining,which proved that NEP1-40-NSCs met the identification criteria of NSC.In the induction differentiation experiment,we got the result that the overexpression of NEP1-40 increased the number of Tuj-1,Oligo2 and MBP positive cells,while the number of GFAP positive cells showed an opposite trend(P<0.05).The same results were obtained in RT-PCR and Western Blot experiments.3.The co-culture of NEP1-40-NSCs and hippocampal tissues found that the axon regeneration area of the NEP1-40-NSCs group was significantly larger than that in the NSCs group(P<0.05);Phalloidine staining showed that NEP1-40 promoted the increase of growth cone area and filopodia length(P<0.05).The detection results of the relative expression of axon regeneration marker protein showed that the expression of GAP-43 and MAP-2 proteins in the NEP1-40-NSCs group was up-regulated,while the expression of APP showed an opposite trend(P<0.05);Compared with the NSCs group,the phosphorylation state of key regulators LIMK1,LIMK2,Cofilin and MLC Ⅱ downstream of the Rho A/ROCK pathway were significantly reduced,and the difference was statistically significant(P<0.05).Conclusion 1.In this study,NEP1-40 genetic-engineered neural stem cells were successfully constructed,and a large amount of NEP1-40 factor expression was detected at the m RNA and proteins level and outside the cell.2.NEP1-40-NSCs can differentiate into neurons and glial cells through in vitro induction,and can the survival rate of rat hippocampal neurons was improved after co-cultivation with it;3.NEP1-40-NSCs can overcome the inhibitory effect of Nogo-66,promote the regeneration of neuronal axons in mature hippocampus which co-cultured with it,and affect the structural development of growth cones.Its mechanism may be through regulating the phosphorylation level of LIM kinase,Cofilin and MLCⅡ to promote axon growth and repair spinal cord injury.