| Transplantation of endothelial progenitor cells can promote endogenous neural stem cells proliferation and differentiation,alleviate inflammation,boost angiogenesis and neuroprotetion.However,researchers indicate that most transplanted cells(such as neural stem cells,bone marrow mesenchymal stem cells,adipose-derived stem cells,induced pluripotent stem cells,olfactory ensheathing cells)died in a few days after transplantation because of the harsh local microenvironment after spinal cord injury.In addition,some scholars believe that the mechanism of transplantation of progenitor cells or stem cells to repair spinal cord injury is through its secreted cytokines,rather than directly differentiated or intergrated into cells.Endothelial progenitor cells(EPCs),mainly obtained from bone marrow or peripheral blood,can secret a variety of growth factors such as HGF,IL-8,PDGF,SDF-1,VEGF and BDNF.Therefore,conditioned medium containing a variety of cytokines secreted by endothelial progenitor cells provide a new way for treatment of spinal cord injury.The aim of this study is to evaluate the effect of endothelial progenitor cells conditioned medium on spinal cord injury and its mechanism.The study was divided into three parts.In the first experiment,bone marrow mononuclear cells were isolated from 4-week-old female Sprague-Dawley rat by density gradient centrifugation and incubated on the culture dishes which coated with fibronectin in endothelial cell growth medium.To produce endothelial progenitor cells conditioned medium(EPC-CM),endothelial progenitor cells were cultured for 48 hours under hypoxic conditions in serum and growth factor-free endothelial cell basal medium,after wipe off cell debris and concentration,the medium was considered as endothelial progenitor cells conditioned medium.Growth factor and serum free endothelial cell basal medium served as control medium(Con-M).VEGF,BDNF,HGF were detected in endothelial progenitor cells conditioned medium and control medium through ELISA.In the second part,we first primary culture bone marrow derived macrophages and spinal cord neurons.Macrophages(M0)were stimulated by lipopolysaccharide to classically activated macrophages(M1),after cultured in endothelial progenitor cells conditioned medium and control medium for 48 hours,classical activated macrophages(M1)and alternatively activiated macrophages(M2)were measured by flow cytometry,in addition,inflammatory cytokines such as IL-1β,IL-6 and IL-10 m RNA expression level were measured by PCR.Spinal cord neurons were cultured by endothelial progenitor cells conditioned medium and control medium for 24 hours then treated by H2O2 for 30 minutes,PI and Hochest33258 double staining were performed.The percentage of apoptotic cells was calculated.Rat aortic rings were cultured in endothelial progenitor cells conditioned medium and control medium for 7 days,the length of microvessels was calculated.Results suggest that endothelial progenitor cells conditioned medium alleviate inflammation,promote angiogenesis and neuroprotection in vitro.In the third part,30 adult female Sprague-Dawley rats(200g-250g)were randomly assigned into 3 groups: EPC-CM group,Con-M group and PBS group,10 rats in each group.Spinal cord was injured using a modified Allen method at T10 level.One milliliter endothelial progenitor cells conditioned medium,control medium,or PBS was injected intraperitoneally per rat daily until the rats were sacrificed.Inflammatory macrophages(CD86+,CD206+),blood vessel endothelium(CD31+)immunostaining and TUNEL assay are carried out to estimate endothelial progenitor cells conditioned medium effect on inflammation,angiogenesis and neuroprotection in vivo.Results showed that endothelial progenitor cells conditioned medium reduce the number of CD86+cells and apoptotic cells,increase the number of CD206+cells and CD31+ lumens,also,NF200 positive density and BBB score were significantly higher in endothelial progenitor cells conditioned medium group.These results suggest that endothelial progenitor cells conditioned medium alleviate inflammation,promote angiogenesis and neuroprotection to promote functional recovery.In summary,endothelial progenitor cells conditioned medium can promote functional recovery in rats with spinal cord injury by inhibiting inflammattion,promoting angiogenesis and neuroprotection. |
