| Objective:The peripheral nervous system(PNS)has a certain regenerative capacity,and the regenerative process that occurs after peripheral nervous injury(PNI)is related to the biological activity in the dorsal root ganglia(DRG).The regeneration process involves cellular regulation and gene expression changes,in which transcription factors play a crucial role,and they can promote cell differentiation,dedifferentiation and transdifferentiation.The aim of this study was to screen the transcription factor Sox9 in mice sciatic nerve injury(SNI)dorsal root ganglia satellite glial cells(DRG-SGCs)by bioinformatics and to verify the expression of Sox9 in DRG-SGCs.Sox9-mediated plasticity changes in DRG-SGCs were initially explored by lentiviral transfection technique.mi RNA sequencing was performed to analyze the mi RNA expression profile of Sox9-mediated plasticity changes in DRG-SGCs.Finally,Sox9-mediated DRG-SGCs were injected into the mice SNI model to observe the repair role of cells in SNI.Methods:1.C57BL/6 mice SNI model were established,m RNA transcriptome sequencing was performed on DRG tissues,and the m RNA-related dataset of DRG-SGCs in the mice SNI model was downloaded using the GEO public database,and the core gene Sox9 was obtained by joint analysis,and Sox9 was further validated in DRG-SGCs at the m RNA and protein levels.2.Immunofluorescence staining to detect the expression of neural stem cell markers Nestin and p75 NTR in DRG-SGCs,and flow cytometry to detect the expression of surface stem cell antigens CD133,CD34,CD45 in DRG-SGCs to initially explore the plasticity of DRG-SGCs.3.Sox9 knockdown stable-transfected DRG-SGCs were constructed and transfection efficiency was detected by real-time quantitative polymerase chain reaction(q RT-PCR).The DRG-SGCs with knockdown of Sox9 were cultured,and the morphological changes of the cells were observed by transmission electron microscopy.The expression of the neural stem/progenitor cell markers Nestin and Sox2 was detected by immunofluorescence staining,and the cells were induced to directional differentiation by different volume fractions of fetal bovine serum(FBS)to explore the effect of Sox9 on the effect of DRG-SGCs on plasticity.4.The survival rate and cell cycle changes of DRG-SGCs with knockdown of Sox9 were detected by CCK8 and flow cytometry,respectively.q RT-PCR was performed in DRG-SGCs with knockdown of Sox9 to detect the expression of transcription factors associated with neuronal transdifferentiation;meanwhile,the Maestro multi-well plate microelectrode array platform was used to observe DRG-SGCs with knockdown of Sox9 action potential changes were observed on the Maestro multi-well plate microelectrode array platform to further explore the effect of Sox9 on the plasticity of DRG-SGCs.5.mi RNA sequencing of DRG-SGCs with knockdown Sox9,and verify the differential mi RNAs and their target genes by q RT-PCR and fluorophore reporter gene assay.6.Single cell suspensions of DRG-SGCs with knockdown of Sox9 were prepared and injected directly at the SNI in mice at the optimal dose,and cell treatment was assessed by sciatic nerve function index(SNFI),Tarlov score,sciatic nerve stem potential amplitude,and gastrocnemius wet weight;Masson staining of sciatic nerve To further observe the repair effect of cells,q RT-PCR was performed to detect the expression of inflammatory factors,neurotrophic factors and nerve growth factors in DRG tissues after cell treatment;Western Blot was performed to detect the expression of growth associated protein 43(GAP 43)in DRG tissues after cell treatment.Results:1.The common core gene Sox9 was obtained by m RNA transcriptome sequencing analysis of DRG-SGCs and DRG tissues after mice SNI.m RNA and protein expression levels of Sox9 were upregulated in DRG tissues and DRG-SGCs after SNI compared to the Sham group,consistent with the sequencing results.2.Immunofluorescence staining detected expression of the neural stem cell markers Nestin and p75 NTR in Sham/SNI DRG-SGCs;Nestin and p75 NTR remained consistently expressed in DRG up to 14 d after SNI.The expression of neural stem cell antigen CD133 was detected on the surface of DRG-SGCs by flow cytometry,and the expression rate increased after SNI compared with the Sham group.DRG-SGCs are a class of cells with plasticity characteristics.3.The Sox9 knockdown stable-transfected DRG-SGCs were constructed,and the best transfection efficiency of "sh-Sox9-1" was detected by q RT-PCR.Sham/SNI DRG-SGCs with knockdown of Sox9 were cultured separately,and sphere-like cell aggregation was observed in the passaged SNI sh-Sox9 DRG-SGCs under white light inverted microscopy,and immunofluorescence staining detected that the passaged SNI sh-Sox9 DRG-SGCs could be labeled with Nestin and Sox2;transmission electron microscopy observed that the passaged SNI sh-Sox9 DRG-SGCs had few organelles,abundant ribosomes,good cell viability and low maturation;immunofluorescence staining detected that 1% volume fraction of FBS could induce differentiation of SNI sh-Sox9 DRG-SGCs into microtubule-associated protein 2(MAP2)positive cells,and 10% volume fraction of FBS induced SNI sh-Sox9DRG-SGCs to differentiate into glutamine synthetase(GS)positive cells,suggesting that SNI sh-Sox9 DRG-SGCs might have stem cell-like characteristics.4.CCK8 assay results showed higher cell survival in SNI sh-Sox9 DRG-SGCs compared to Sham sh-Sox9 DRG-SGCs.flow cytometry detected a decrease in the percentage of cells in G0G1 phase in SNI sh-Sox9 DRG-SGCs compared to Sham sh-Sox9 DRG-SGCs,and an increase in the percentage of cells in S phase compared to Sham sh-Sox9 DRG-SGCs.q RT-PCR detected an upregulation of m RNA expression of transcription factors Brn3 a,Ngn1,and Ngn2 associated with neuronal transdifferentiation in SNI sh-Sox9 DRG-SGCs compared to Sham sh-Sox9DRG-SGCs;Maestro multi-well plate microelectrode array platform recorded the appearance of early neuron-like firing process in the SNI sh-Sox9 DRG-SGCs,and it was speculated that SNI sh-Sox9 DRG-SGCs might show transdifferentiation phenomenon.5.After mi RNA sequencing of DRG-SGCs with low Sox9 knockdown,the known differential mi RNAs analyzed were mi RNA-381-3p,mi RNA-5108,mi RNA-5119,mi RNA-5126,mi RNA-6240;among them,mi RNA-381-3p was reported to have a key role in proliferation and differentiation of neural stem cells in vitro;q RT-PCR detected the upregulation of Ebf3,a target gene possibly associated with mi RNA-381-3p,Ebf3 was reported to have a key role in proliferation and differentiation of bone marrow mesenchymal stem cells in vitro.6.The optimal dose of SNI sh-Sox9 DRG-SGCs single cell suspension was injected directly into mice SNI,SNFI values,Tarlov score,sciatic nerve stem potential values and gastrocnemius wet weight,Masson staining of sciatic nerve suggest that SNI sh-Sox9 DRG-SGCs treated mice SNI 2w-4w,the damage was repaired to some extent;After SNI sh-Sox9 DRG-SGCs treatment of mice SNI,q RT-PCR results showed that the m RNA expression of inflammatory factors TNF-α,IL-6 and IL-1β were down-regulated,and the m RNA expression of neurotrophic factor BDNF and nerve growth factor NGF were up-regulated in the SNI-Saline group compared with the SNI-Saline group;Western Blot showed that the expression of GAP43 protein was increased compared with the SNI-Saline group.The results suggest that SNI sh-Sox9 DRG-SGCs have a restorative effect on mice SNI.Conclusions:1.m RNA and protein expression levels of Sox9 were up-regulated in plastic cell DRG-SGCs after SNI.2.Knockdown of Sox9,SNI DRG-SGCs showed stem cell-like plasticity characteristics and transdifferentiation,and mi RNA sequencing analysis speculated that mi RNA-381-3P was downregulated and Ebf3 was up-regulated during this process.3.Single cell suspensions of SNI DRG-SGCs with knockdown of Sox9 had some repairing effect on mice SNI. |
PDF Full Text Request