| Background and ObjectiveSpinal cord injury(SCI)is a spinal cord or cauda equina injury caused by trauma,ischemia,infection,immunity,tumor,drinking and other factors.Often cause paralysis,sensory abnormalities,urinary disorders,neuralgia,dyspnea,sexual dysfunction and other temporary or permanent injury below the segment of nerve dysfunction.Worldwide,since 1995,the incidence of spinal cord injury ranges from 10.4 / million to 83 / million per year,and there are about 60000 new cases of spinal cord injury in China every year.Among them,the vast majority of patients with spinal cord injury are caused by traffic accidents,high-altitude falls,violent injuries and other injuries,and such traumatic spinal cord injuries often leave permanent neurological dysfunction,resulting in a very high rate of disability.The happiness of personal and family life,as well as the tranquility and prosperity of the country and society,have been brought a great burden.All over the world,major scientific research institutes and medical institutions are also using a lot of manpower and material resources to continuously invest in spinal cord injury related research,in order to find more effective treatment methods to help more patients recover to health.Among the many promising research directions,stem cell-related treatment is undoubtedly the star to help patients with spinal cord injury to reduce acute damage and promote the recovery of neural function,which has great research value and potential.Mesenchymal stem cells(MSCs),has been used in many experimental treatments of stem cell transplantation for spinal cord injury,which has a variety of sources,and is easy to obtain,easy to culture,can secrete a variety of protective neurotrophic factors and has a certain differentiation ability,As one of the most widely used stem cells,it has also achieved certain results in the treatment process.However,direct transplantation of MSCs still has some disadvantages,such as short survival time,difficult to reach the damaged area,rejection,allergic reaction,carcinogenesis,abnormal differentiation,difficult to reuse a large number of and so on.Other studies have shown that the paracrine effect of transplanted stem cells plays a dominant role in its therapeutic function,but the number of neurons and glial cells differentiated into neurons and glial cells is very limited.Therefore,the application of homogeneous vesicles secreted by MSC,which contain a variety of DNA,RNA,protein and neurotrophic factors about 40~100nm in diameter,that is,the exocrine body derived from MSC,can not only avoid the disadvantages of cell transplantation,but also avoid the disadvantages of cell transplantation.It can also regulate,screen and mass produce exosomes with specific functions according to the needs.Therefore,the application of MSC-derived exocrine body in the treatment of spinal cord injury has a broader prospect,but also has greater scientific research and clinical value.Pericyte(Pericyte,PC)plays a key role in many physiological and pathophysiological processes,and it is of great significance to the systemic vascular stability of various organs.Such as the blood-brain barrier(BBB)of the central nervous system,the blood-spinal cord barrier(BSCB)and the branches of the coronary artery,and more importantly,pericytes and microvascular endothelial cells,glial cells and neurons of the central nervous system constitute neurovascular units.Pericytes regulate and maintain the normal function of the blood-brain barrier by regulating the diameter of capillaries and microvascular perfusion in the central nervous system.The absence of pericytes in the central nervous system will lead to the destruction of the blood-brain barrier,neuroinflammation and the formation of aneurysms.In addition,some studies have shown that pericytes have the function of secreting growth factors and nutritional factors to peripheral cells and cell matrix.In spinal cord injury,the damage,deletion and migration of spinal cord microvascular pericytes will cause the destruction of blood-spinal cord barrier and aggravate inflammation and edema,and its abnormal differentiation may aggravate scar formation and affect the prognosis.Its abnormal paracrine function may increase the permeability of blood-spinal cord barrier.Therefore,pericytes play a key role in spinal cord injury and central nervous system diseases.however,the understanding of pericytes is still relatively shallow,and there is relatively little research on its function and mechanism,which needs to be further explored.It is expected to become a new target for the treatment of many diseases.In this study,bone marrow mesenchymal stem cell derived exosome(BMSC-exo)was extracted by kit method and injected in situ into the injured site of SCI rat model.The effects of transplanted exosomes on pericyte coverage,stability of blood-brain barrier and recovery of nerve function were observed histologically and ethologically.In the cell model in vitro,the effect of exocrine therapy on endothelial barrier was observed,and its potential molecular mechanism was explored.Materials and methods:BMSCs were isolated by whole bone marrow culture and cultured and subcultured under suitable conditions.the exocrine bodies were extracted by exosome extraction kit.the exosomes were identified by transmission electron microscopy(TEM),particle size analysis and flow cytometry(FCM).The exocrine body was injected in situ into SCI rats.after gait analysis at 7 days,the samples were taken for immunofluorescence staining to observe PDGFR?,CD31,NF?B and other,evens blue to detect blood-brain barrier permeability,western blot for the determination of tight junction protein in tissue homogenate.The,Luminex assay of primary pericytes of rat spinal cord treated with TNF ? was used to detect the effect of exocrine on the secretion of cytokines by pericytes.The supernatant of pericyte culture medium was added to rat spinal cord vascular endothelial cells to observe the effect of pericyte medium supernatant on endothelial barrier.Results1.Bone marrow mesenchymal stem cells extracted from rat bone marrow were detected to be CD90+,CD34-by western blot.2.The exocrine bodies isolated from rat bone marrow mesenchymal stem cells showed a relatively uniform round or circular model structure with a diameter of about 40-100 nm under electron microscope,and western blot showed CD9+,CD63+.3.On the 7th day after operation,BBB score and gait analysis showed that the recovery of hindlimb motor function in the treatment group was better than that in the injury group,and there was significant difference between the two groups.4.The spinal cord specimens taken 7 days after operation showed that the permeability of blood-spinal cord barrier was lower,the level of endothelial tight junction protein was higher,and the pericyte coverage was higher in the treatment group than that in the injury group,and the differences was statistically significant.5.The primary spinal cord vascular pericytes extracted from rat spinal cord were stained with immune sunlight for ?-SMA+,PDGFR?+,and immunofluorescence showed that PKH26 labeled exosomes entered pericytes.6.The supernatant of pericyte culture medium pretreated with TNF ? had higher VEGF level than that of the control group.when it was added to rat vascular endothelial cells,the endothelial barrier function was decreased compared with the control group,and the difference was statistically significant.ConclusionBone marrow derived mesenchymal stem cell exosomes can reduce functional impairment and promote restoration in spinal cord injury rat model by affect pericyte activation and paracrine of traumatic area. |
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