The Research On Regulatory Effects Of Rat Dermal Papilla Cells On M1/M2 Macrophage Polarization

Research background and purposeSpinal cord injury(SCI)is one of the most serious complications of spinal cord injury,and is one of the worst prognosis diseases in the world.The spinal cord is an important part of the central nervous system,is the centre of many reflection,spinal cord injury often leads to severe dysfunction of the lower limbs,to the patient and family and society a heavy economic burden,therefore,for the treatment of spinal cord injury has become a major issue in medical field.However,due to the lack of regeneration ability of neurons,traditional drugs and surgical treatment to promote nerve repair is very limited.In contrast,cell transplantation for the treatment of spinal cord injury may complement damaged neurons,but early inflammatory responses in spinal cord injury often result in the death of transplanted cells.On the one hand,the inflammatory response has a devastating effect on the death of glial cells,neurons,and transplanted cells,on the other hand,inflammation clears the necrotic cell debris and is beneficial to nerve regeneration and functional repair.How to understand the two sides of inflammation and improve the adverse microenvironment after injury is the key to the cell transplantation treatment of spinal cord injury.It has been proved that the dual effects of inflammation on the promotion of injury and repair are related to the polarization of macrophages.Macrophages,as the most important phagocytic and antigen presenting cells in vivo,show different functions in different microenvironment.The process of dynamic change of macrophage function with the change of microenvironment is called macrophage polarization.The most closely related to the repair of injury were the "classical activated" macrophages(type M1)and "alternative activated macrophages"(type M2).M1 type macrophages secrete IL-1?,IL-6,TNF-? and other pro-inflammatory factor,active oxygen and nitric oxide,caused further damage,inhibition of axon regeneration.The M2 type macrophages secrete IL-4,IL-10,TGF-? and other anti-inflammatory factors and neurotrophic factor NGF and so on,play a neuroprotective role,promote the repair of damaged nerve and axon regeneration.Therefore,M1 type and M2 type macrophage ratio is an important factor in determining the outcome of spinal cord injury,promote M1 macrophages transforming to M2 contribute to the improvement of spinal cord injury against the inflammatory microenvironment,is conducive to the treatment and prognosis of spinal cord injury.Stem cells are a kind of pluripotent cells which have the ability of self replication.They are often used as seed cells for cell therapy.The research report of embryonic stem cells(ES),hematopoietic stem cells(HSCs),neural precursor cells,mesenchymal stem cells(MSCs)have potential for the treatment of spinal cord injury,but,embryonic stem cells and hematopoietic stem cells are difficult to obtain and there are ethical issues,Hematopoietic stem cells often cause graft rejection,and typical MSCs,bone marrow mesenchymal stem cells(BMSCs)into nerve ability is controversial,therefore need to find new sources of ideal seed cells.Dermal papilla cells(DPCs)are easy to obtain and abundant in origin,and there are skin derived precursor cells(SKPs)in the craniofacial papilla,which have the characteristics of neural crest stem cells and have the potential of nerve differentiation.It expresses the same surface marker with MSCs,which suggests that it may have the same immunomodulatory properties as MSCs.The previous study found that in the acute phase of spinal cord injury,DPCs and BMSCs were transplanted into the rats spinal cord injury model,DPCs better inhibiting inflammatory reaction of death than BMSCs,the number of DPCs in the site of spinal cord injury was significantly higher than that in BMSCs,and the infiltration of macrophages in the DPCs transplantation group was significantly more than that in BMSCs.RT-PCR confirmed that DPCs expressed IL-10,TGF-? and VEGF at m RNA level,and all of these genes could promote the polarization of type M2 macrophages.These results suggest that DPCs may play a regulatory role in macrophage polarization.Therefore,the purpose of this study is to to obtain the purified rat dermal papilla cells,based on identification of surface markers and differentiation potential,explore its regulation of macrophage polarization,to open up a new source of seed cells in cell therapy for spinal cord injury.And the BMSCs was used as the positive control.Method 1.BMSCs and DPCs were isolated and cultured.The expression of surface markers was detected by flow cytometry,and the expression levels of M1/M2 and macrophage related cytokines were detected by real-time quantitative PCR.2.Bone marrow derived macrophages(BMDMs)were isolated and cultured,and their purity was identified by immunofluorescence,flow cytometry and special staining,and then induced by LPS+ IFN-? stimulation to form M1 macrophage.3.BMSCs and DPCs were co cultured with BMDMs for 24 hours respectively,detect the changes of the expression levels of M1,M2 markers and the expression of M1/M2 related cytokines in BMDMs macrophages.Result 1.In this experiment,DPCs from rat cranial facial whiskers and BMSCs were successfully isolated and cultured.The DPCs has the same mesenchymal stem cell markers and multilineage differentiation potential as BMSCs.2.Compared with BMSCs,DPCs is high expression of M2 type macrophage related cytokines IL-4,IL-10,VEGFA,while low expression of M1 type macrophage associated cytokines IL-1?,IL-6,TNF-?.3.BMDMs were successfully isolated and cultured,and were induced into type M1 macrophages.4.After co cultured with DPCs,M1 type macrophages decreased,the markers i NOS,CD86 and related cytokines TNF-? expression level decreased,while M2 type macrophages increased,the expression level of the markers CD206 and related cytokines IL-10,VEGFA also increased.And the changes than BMSCs co culture group significantly.ConclusionDPCs has the same mesenchymal stem cell markers and differentiation potential with BMSCs,high expression of M2 type macrophage associated factor,stronger ability to regulate M1 type macrophages to M2 type macrophages than BMSCs cells,in treatment of spinal cord injury is expected to become a new source of seed cells.