| Objective: Spinal cord injury is one of the more serious diseases in spine surgery.It is caused by physical damage.After the injury,the patient often has distant limb sensation and motor dysfunction.Spinal cord injury can be divided into incomplete or complete nerve damage,depending on the type and severity of the injury,the length of time required for neurological recovery varies.Because it is difficult to repair itself after nerve injury,people with severe damage often have residual limb dysfunction for life.This not only reduces the quality of life of the patient,but also the costs associated with care and treatment imposes greater economic pressure on the patient's family.At present,the incidence of spinal cord injury is gradually increasing.The nerve repair and regeneration after spinal cord injury has become a serious challenge for all researchers.With the rapid development of modern biotechnology,people gradually realize that nerve cells can be regenerated by transplantation of stem cells,and the nerve function of the damage can be repaired.Neural stem cells are members of stem cells and are characterized by proliferation,self-renewal,and differentiation into a variety of neural tissue cells.Neural stem cells can be divided into endogenous and exogenous neural stem cells.Endogenous neural stem cells are less abundant in the adult central nervous system and have lower regenerative capacity.By stimulating endogenous neural stem cells,it is not enough to compensate for the loss of nerve cells and tissues.Currently,compensation for the loss of nerve cells and tissues by neural stem cell transplantation has become one of the treatment methods for spinal cord injury.However,while neural stem cells continue to receive attention from the academic community,they also face many problems.For example,the safety of stem cell transplantation(tumorigenicity)and the low proportion of neuronal differentiation after stem cell transplantation cannot replace the necrotic nerve cells in the damaged area.Therefore,it is necessary to optimize the treatment of neural stem cells before cell transplantation,thereby improving the safety and effectiveness of cell transplantation.Micro RNAs are widely present in the central nervous system as small non-coding RNAs,which have been shown to be involved in the proliferation and differentiation of neural stem cells.The genetic regulation of neural stem cells by micro RNAs to improve their proliferation and differentiation activities is an optional optimization.The main purpose of this paper is to screen micro RNAs(mi R-329-3p)differentially expressed between neural stem cells and neurons by gene chip,and further study its role and related molecular mechanisms in the proliferation and differentiation of neural stem cells.Thus creating favorable conditions for neural stem cell transplantation to repair nerve damage.Methods: This experimental study was divided into two parts.In the first part,SD rat neural stem cells were successfully cultured in vitro,and the growth state of the neurospheres and the cell morphology changes after induction of differentiation were observed by microscopy.Using gene chip to find mi R-329-3p differentially expressed between neural stem cells and neurons,the expression of mi R-329-3p was detected by RT-q PCR at different time points of differentiation of neural stem cells,and the chip results were verified.In the second part,mi R-329-3p was transfected into neural stem cells,and the expression of mi R-329-3p was detected by fluorescence microscopy and RT-q PCR.The proliferation of neural stem cells after transfection of mi R-329-3p was detected by CCK-8,Ed U and PCNA methods.The mi R-329-3p target gene was predicted from the Target Scan and mi RDB sites,and the target gene E2F1 was verified using a dual luciferase assay.In order to further investigate the targeted regulation of mi R-329-3p on E2F1 in neural stem cells,the expression of E2F1 was detected by RT-q PCR and Western blot after overexpression of mi R-329-3p.E2F1 si RNA was used to interfere with E2F1 expression,and the expression of E2F1 was detected by RT-q PCR and Western blot.The proliferation of neural stem cells after si RNA interference was detected by CCK-8 and Ed U methods.In order to verify that mi R-329-3p regulates the proliferation of neural stem cells through E2F1,the mi R-329-3p mimic and E2F1 overexpression plasmids or control plasmids were co-transfected into neural stem cells,and the proliferation of neural stem cells was further detected by CCK-8 and Ed U methods.In the study of the effect of mi R-329-3p on the differentiation of neural stem cells,we overexpressed mi R-329-3p in neural stem cells,and detected the expression of neuronal marker Tuj1 after 3 days of differentiation by RT-q PCR,Western blot and cellular immunofluorescence.The expression of E2F1 in the differentiation of neural stem cells was detected by RT-q PCR and Western blot.The mi R-329-3p mimic and E2F1 overexpression plasmids or control plasmids were co-transfected into neural stem cells.The expression of neuronal marker Tuj1 was detected by RT-q PCR,Western blot and cellular immunofluorescence.Results: The SD rat neural stem cells were observed under an inverted phase contrast microscope and the cells were grown in a growth medium with no adherent differentiation.After 72 hours of differentiation culture,the cell morphology was obviously changed,and the long protrusions were extended,showing neuron-like appearance.A gene chip was used to find mi R-329-3p differentially expressed between neural stem cells and neurons.The expression of mi R-329-3p was gradually increased during the differentiation of neural stem cells by RT-q PCR.Fluorescence microscopy showed that the transfection efficiency of neural stem cells adherently transfected with mi R-329-3p mimic was about 60% after 24 hours.After 48 hours of suspension transfection,the neural stem cells were aggregated into spheres and fluoresced under fluorescence microscope.RT-q PCR revealed that the expression of mi R-329-3p was significantly increased after transfection of mi R-329-3p mimic in neural stem cells.Overexpression of mi R-329-3p inhibited neural stem cell proliferation.E2F1 was identified as a target gene of mi R-329-3p by bioinformatics prediction and using dual luciferase assay.mi R-329-3p inhibited the expression of E2F1 genes and proteins expression in neural stem cells.E2F1 si RNA could effectively interfere with the expression of E2F1,and inhibitied neural stem cell proliferation.The mi R-329-3p mimic and E2F1 overexpression plasmids or the negative control plasmid were co-transfected,and E2F1 overexpression promoted neural stem cell proliferation compared with the control group.In neural stem cell differentiation culture,mi R-329-3p overexpression could promote the expression of the neuronal marker Tuj1 gene and protein expression.Cellular immunofluorescence showed that overexpression of mi R-329-3p promoted the differentiation of neural stem cells into neurons.During the differentiation of neural stem cells,the expression of E2F1 gene and protein expression gradually decreased.The mi R-329-3p mimic and E2F1 overexpression plasmids or the negative control plasmid were co-transfected into neural stem cells,and it was found that the expression of the neuronal marker Tuj1 was decreased in the E2F1 overexpression group compared with the control group.Cellular immunofluorescence results also showed that overexpression of E2F1 inhibited the differentiation of neural stem cells into neurons.Conclusion:(1)mi R-329-3p is differentially expressed in neural stem cells and neurons;mi R-329-3p is gradually increased in neural stem cell differentiation.(2)mi R-329-3p can inhibit the proliferation of neural stem cells and promote their differentiation into neurons.The mechanism of action is related to the inhibition of the expression of target gene E2F1. |
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