Deletion Of KLF4 Promotes Axonal Regeneration In Mammals

Purpose:Axon regeneration is the basis of functional recovery after nerve injury.However,the regeneration of axons,especially the axons of the central nervous system,is still a difficult problem in clinical practice.The integrity of neuron axons plays a very important role in material transport and signal transmission between neurons.The strength of axon regeneration depends on the environment of the neuron and its own regeneration ability.Kruppel-like factor 4(KLF4)is a member of the zinc finger protein transcription factor family.It has been found that Klf4 gene knockout can promote axonal regeneration of retinal ganglion cells,but the effect of Klf4 gene knockout on the regeneration of corticospinal tract and sciatic nerve is unknown.Therefore,this project intends to study the effect of Klf4 knockout on axonal regeneration by means of spinal cord injury model,sciatic nerve injury model and in vitro neuron culture.Method:1.In order to construct Klf4-specific knockout mice in DRG sensory neurons,Klf4f/f mice were used to mate with Advillin-Cre(Avil-Cre)mice and Pirt-Cre mice,and the offspring were then mated with Klf4f/f mice to obtain Klf4f/f/Avil-Cre mice and Klf4f/f/Pirt-Cre mice,DRG tissue sections were taken for immunofluorescence staining of Tuj 1 antibody and KLF4 antibody.In addition,the amount of KLF4 protein was detected by Western Blot.2.Axotomy(sciatic nerve dissection)was performed on adult ICR mice,and the expression changes of KLF4 protein at 1,3,7 and 21 days after axotomy were detected by Western Blot.Then,the changes of KLF4 protein were detected by immunofluorescence staining on DRG tissue sections at 7 days after axotomy.3.DRG sensory neurons of Klf4f/f/Avil-Cre?Klf4f/f/Pirt-Cre and Klf4f/f mice were cultured in vitro,and 3 days later,neuron-specific antibodies were used for immunofluorescence staining to measure the length of neuronal axon.4.The Klf4f/f/Avil-Cre?Klf4f/f/Pirt-Cre and Klf4f/f mice were electrotransferred to EGFP plasmid in vivo to mark sensory neurons.The sciatic nerve was crushed after 2 days,and the sciatic nerve was compressed after another 3 days to measure the length of regenerated axons.5.For Klf4f/f mice,the cerebral cortex was injected with AAV-Cre virus to knock out the cortical Klf4 gene,and the control was AAV-GFP virus.Two weeks later,T8 spinal cord crush was performed.After 6 weeks,the cortical spinal cord tract was labeled with BDA by cerebral cortex injection,and the mouse spinal cord was taken and sectioned for another 2 weeks.The sections were stained with CyTM3 to show CST,and statistics of regenerated axons.Results:1.Compared with Klf4f/f mice,immunofluorescence staining of DRG sections of Klf4f/f/Avil-Cre?Klf4f/f/Pirt-Cre mice showed that there was little KLF4 protein in the nucleus of DRG sensory neurons.Western Blot showed the expression level of KLF4 protein in DRG in Klf4f/f/Avil-Cre(P<0.01)?Klf4f/f/Pirt-Cre(P<0.01)mice was very low,indicating that Klf4-specific knockout mice in DRG sensory neurons were successfully constructed.2.After axotomy in ICR mice,Western Blot showed that the expression level of KLF4 was decreased,which was the lowest on day 7(P<0.01)and slightly increased on day 21 compared with day 7.Immunofluorescence staining of DRG tissue sections at 1 week after axotomy indicated that KLF4 protein in the nucleus was decreased,suggesting that KLF4 might be related to axonal regeneration.3.DRG sensory neurons cultured in vitro showed that the axonal regeneration length of Klf4f/flAvil-Cre(P<0.01)?Klf4f/f/Pirt-Cre(P<0.001)mice was longer than that of Klf4f/f mice.4.Klf4 knockout can promote regeneration after sciatic nerve crush(P<0.001).5.Deletion of the Klf4 gene in the cerebral cortex can promote regeneration of the corticospinal tract(P<0.05).Conclusion:Deletion of Klf4 gene can promote axonal regeneration in mammals.