The Research On Conditioned Medium Of Schwann-like Cells Derived From Human Neural Stem Cells To Promote Angiogenesis And Repair Sciatic Nerve Injury

Objective:To investigate the effects of conditioned medium of Schwann-like cells derived from human neural stem cells（hereinafter referred to as SC-like cell CM）on endothelial cell proliferation,migration and tube formation in vitro,as well as the role of promoting angiogenesis and nerve repair after sciatic nerve crush injury in rats,and explore the possible mechanism of SC-like cell CM to promote angiogenesis.Methods:（1）The expressions of neural markers in human embryonic stem cell derived-neural stem cells（hESC-NSCs）and Schwann-like cells derived from human neural stem cells（hereinafter referred to as SC-like cells）were analyzed by flow cytometry,and the expressions of neurotrophic factors and pro-angiogenic genes were analyzed by RT-qPCR.（2）The effects of SC-like cell CM on human umbilical vein endothelial cells（HUVEC）proliferation,migration,and tube formation were examined by MTT assay,BrdU staining,Transwell migration assay,scratch test and tube formation assay.（3）The effect of SC-like cell CM on PI3K/Akt/mTOR signaling pathway in HUVEC was detected by western blot.（4）The effects of SC-like cell CM on the proliferation,migration and tube formation of HUVEC treated with PI3K inhibitor LY294002 were detected by MTT assay,Transwell migration assay,scratch test and tube formation assay.（5）The model of sciatic nerve crush injury in SD rats was established in vivo.The effect of SC-like cell CM on functional recovery after sciatic nerve crush injury were observed by footprint analysis,sciatic nerve function index（SFI）,wet weight of gastrocnemius muscle and muscle fiber morphology.（6）The effect of SC-like cell CM on myelin regeneration after sciatic nerve crush injury was analyzed by transmission electron microscope（TEM）.（7）The effect of SC-like cell CM on angiogenesis of injured site after sciatic nerve crush injury was analyzed by Evans blue leakage and CD34 immunofluorescence staining.Results:（1）Flow cytometry results showed that compared with hESC-NSCs,Nestin protein level decreased in SC-like cells,while GFAP and P75^NTR protein level increased.RT-qPCR showed that compared with hESC-NSCs,the expressions of neurotrophic factors（BDNF,NGF and NT-3）and angiogenic molecules（VEGF-A,Ang-1 and Ang-2）were up-regulated in SC-like cells（all P<0.05）.（2）After treatment with SC-like cell CM,it significantly promoted HUVEC proliferation,migration,and tube formation,which were statistically significant compared with the control group（all P<0.05）.（3）After treatment with SC-like cell CM,the expressions of p-Akt and p-mTOR increased in HUVEC.And the increase of the expressions of p-Akt and p-mTOR caused by SC-like cell CM could effectively reverse after treatment with PI3K inhibitor LY294002.（4）Treatment with PI3K inhibitor LY294002 can effectively reverse the changes of HUVEC proliferation,migration and tube formation induced by SC-like cell CM.（5）Compared with the control group,the SC-like cell CM treatment significantly increased SFI and improved the gastrocnemius atrophy at 14 days after sciatic nerve crush injury（all P<0.05）.（6）Compared with the control group,the number of myelinated fibers and the thickness of myelin sheath in the SC-like cell CM group were significantly increased at 14 days after sciatic nerve crush injury（all P<0.05）.（7）Compared with the control group,the SC-like cell CM treatment significantly reduced vascular permeability and increased microvessel density at 14 days after sciatic nerve crush injury（all P<0.05）.Conclusions:（1）SC-like cell CM has the effect to promote angiogenesis in vitro.（2）SC-like cell CM exert pro-angiogenic property by activating PI3K/Akt/mTOR signaling pathway.（3）SC-like cell CM promotes myelin regeneration and nerve function recovery after sciatic nerve crush injury in vivo.（4）SC-like cell CM promotes angiogenesis after sciatic nerve crush injury in vivo.