| BackgroundSpinal cord injury（SCI）is a serious traumatic disease that occurs in the central nervous system（CNS）.The disability rate is high,and the incidence rate is increasing year by year,which brings a heavy burden to patients,families and society.For centuries,the clinical use of drugs,surgical intervention,hyperbaric oxygen,physical therapy and other methods to treat SCI and its sequelae have failed to achieve good results.Therefore,finding more effective treatment methods and targets has become an urgent problem to be solved.Animal experiments have shown that inhibiting a certain complement component can reduce inflammation and neuronal damage after SCI,and promote nerve regeneration and functional recovery.Knockout of the C 3 gene can reduce secondary damage after SCI in mice,inhibit early inflammation and improve nerve regeneration.The use of C5a antagonists can significantly improve the recovery ability of mice after SCI,and improve the histology of the spinal cord of mice after SCI.At present,glucocorticoids（methylprednisolone,etc.）in the guidelines for the treatment of acute SCI are routinely used in clinical patients with severe SCI,and as immunosuppressants,its side effects and adverse reactions also limit its clinical application.Therefore,the development of new immunomodulators to improve the changes of the immune microenvironment after SCI has important clinical significance.In recent years,preclinical studies have found that Bone Marrow Mesenchymal Stem Cells（BMSCs）transplantation plays a role in nerve repair in animal models of SCI,and is considered to be the most promising treatment strategy for SCI.However,the teratogenicity,immune rejection,pulmonary embolism,low transplantation rate,and low survival rate of stem cell transplantation limit its clinical application.Current research has found that transplanted BMSCs release a variety of biologically active substances in the host,such as neurotrophic factors,extracellular vesicles and immunomodulatory substances.These biologically active substances can communicate with host cells through paracrine effects and change the behavior of cells to play a role in neuroprotection and repair.Exosomes（Exo）are membranous lipid small extracellular vesicles with a diameter of about 30-150 nm,which can be produced by various tissues and cells.Exosomes are rich in biologically active substances:proteins,lipids,mRNA,microRNA（miRNA）and long non-coding RNA.In recent years,studies have found that exosomes have a variety of functions,which can improve ischemia-reperfusion injury,inhibit lung inflammation,promote angiogenesis,promote wound healing,and repair brain nerve cells.This topic uses the hemisection model of SCI in rats to explore the protective effect of exosomes derived from bone mesenchymal stem cells（BMSCs-Exo）on injured spinal cord tissue after SCI,and explores the effects of changes in the complement system on this process.It provides a new idea for the treatment of SCI,and provides a theoretical and practical basis for finding a safer,more effective,cell-free treatment of SCI.Part Ⅰ Extraction and Identification of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells and in Vitro Endocytosis ExperimentsObjective:To isolate,extract and identify exosomes derived from BMSCs,and determine that they can be absorbed by cells through exosomes tracing technology,so as to provide new treatment strategies for the treatment of SCI.Methods:Primary isolation and culture of rats BMSCs,the conditioned medium was collected,and the exosomes derived from BMSCs was extracted through Millipore ultrafiltration tube ultrafiltration combined with exosome extraction kit qEV separation column,dynamic observation of BMSCs-Exo by Transmission Electron Microscopy（TEM）,analysis of its size and concentration by nano-tracking analysis technology,Western blotting to identify the marker protein molecules of BMSCs-Exo;PKH26 labeling BMSCs-Exo were incubated in BV-2 cells for 12 hours,and the distribution of BMSCs-Exo was detected by immunofluorescence.Results:BMSCs-Exo can present round or round-like single vesicles or multiple vesicles under TEM.The results of nanoparticle tracking analysis showed that the particle size was 81.2±4.2 nm.Western blotting confirmed that it expressed CD9,TSG101 characteristic surface molecule;immunofluorescence results show that PKH26 labeled BMSCs-Exo can be endocytosed by BV-2 cells.Conclusion:1.Ultrafiltration combined with size exclusion chromatography can extract BMSCs-derived exosomes from the cell-based supernatant of bone marrow mesenchymal stem cells,and can increase the enrichment of exosomes.2.The exosomes extracted from the BMSCs cell-based supernatant meets relevant international standards in terms of size,morphology,and characteristic proteins.3.BMSCs-Exo can be taken up by microglia.Part Two:Study on the Effect of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells on the Functional Recovery of Rats after SCIObjective:The effects of BMSCS-Exo on spinal cord injury recovery and functional behavior of rats were investigated by establishing SCI model of rats.Methods:1.Establishment of rat SCI experimental modelTo study SCI,we used the spinal cord hemisection method to construct the SCI model,using male Wistar rats weighing 200-250g.After the rats were anesthetized,the T9 and T10 spinous processes and lamina were removed,the white spinal cord and the central blood vessels on the dorsal side of the spinal cord were exposed,and the right spinal cord was cut with an ophthalmic iris knife to complete the model construction.2.Animal grouping and administrationExperimental animals were randomly divided into 3 groups of sham operation group（Sham group）,spinal cord injury group（SCI group）and exosomes treatment group（SCI+Exo group）.It was administered by tail vein injection.The SCI+Exo group was given 500μL BMSCs-Exo each.The Sham group and SCI group were given the same volume of Phosphate buffered saline（PBS）.3.The effect of BMSCs-Exo on the motor function of ratsThe Basso,Beattie and Bresnahan scores（BBB）and the inclined plate test were used to observe and record and monitor the motor function on 1,3,7,14,21 and 28 days respectively to evaluate the effect of BMSCs-Exo on the rat’s movement functional impact.4.BMSCs-Exo improves the histological detection of SCI in ratsThe paraffin sections of the spinal cord of rats were prepared by perfusion and fixation at 3 and 28 days after modeling,and routine hematoxylin-eosin（HE）staining and neuronal specific nuclear protein（NeuN）immunohistochemical staining were performed to evaluate the spinal cord tissue condition.5.BMSCs-Exo fluorescent staining tracesRats were injected with PKH67-labeled BMSCs-Exo through the tail vein to detect the distribution of BMSCs-Exo in vivo and whether it was accepted by the spinal cord tissue.Results:1.BMSCs-Exo can alleviate spinal cord injury and improve the recovery of motor function in ratsThe BBB score was used to evaluate the recovery of motor function of rats after modeling.Before the experiment,all rats had BBB scores of 21 points.All rats after SCI modeling showed paralysis of hind limbs.During the observation period,the BBB scores of the SCI group and SCI+Exo group gradually increased.After entering the 7th day,it was observed that the motor function score of the SCI+Exo group was significantly better than that of the SCI group（p<0.05 on day 7;p<0.01 on day 14;p<0.05 on day 21;p<0.05 on day 28）.The oblique plate experiment was used to evaluate the muscle strength recovery of rats after modeling.Before the experiment,the angle of the oblique plate of all rats was 70°,and the rats after SCI modeling showed paralysis of hind limbs.During the observation period,the SCI group and SCI+Exo group,the rats in the inclined plate experiment gradually increased.After entering the 7th day,it was observed that the motor function score of the SCI+Exo group was significantly better than that of the SCI group（p<0.05 on day 7;p<0.05 on day 14;p<0.05 on day 21;p<0.05 on day 28）.2.BMSCs-Exo can reduce the pathological damage of spinal cord tissue after SCIHE staining and immunohistochemical staining found that the Sham group cells have normal morphology,orderly arrangement,clear nuclei,and clear white matter boundaries;SCI group cells can see more and larger damaged cavities,the structure is obviously loose,and some cells have obvious vacuoles.The nuclei are deeply stained,and the cell morphology in the damaged area is poor,with more cell debris scattered.After treatment with BMSCs-Exo,the morphological structure of cells in the SCI+Exo group was improved compared with that in the injured group,with fewer vacuoles and fewer inflammatory cells.3.BMSCs-Exo injected into the tail vein can be absorbed by rats and enter the spinal cord tissueThe PKH67 fluorescently labeled BMSCs-Exo tracing experiment showed that the fluorescent signal of BMSCs-Exo can enter the spinal cord tissue and is also distributed in the liver,spleen,and kidney.Fluorescence microscopy observed that a fluorescent signal appeared at the injured site one day after SCI,the fluorescence intensity increased significantly after 3 days,and decreased significantly after 7 days.The distribution in liver,kidney,and spleen tissues showed that the fluorescence intensity was strongest on day 1,weakened on day 3,and weakest on day 7.Conclusion:1.BMSCs-Exo can reduce spinal cord injury and improve motor function recovery.2.BMSCs-Exo can reduce the pathological damage of spinal cord tissue after SCI.3.BMSCs-Exo can reach the injury site on the first day after tail vein injection,and the amount of BMSCs-Exo reaches the maximum after 3 days.Part Ⅲ Proteomic Analysis of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells in Rats after SCI and Study on the Mechanism of Inhibiting Complement ActivationObjective:Differential proteins in the microenvironment of spinal cord injury after SCI were screened by proteomics techniques,and their functions were annotated and enriched.The selected complement proteins were further analyzed.The regulation mechanism of BMSCS-Exo in SCI treatment at the protein molecular level was preliminarily discussed,which provided a theoretical basis for further optimization of BMSCS-Exo in SCI treatment.Methods:The experimental animals were randomly divided into 3 groups of sham operation group（Sham group）,spinal cord injury group（SCI group）and exosomes treatment group（SCI+Exo group）.It was administered by tail vein injection.The SCI+Exo group was given 500μL BMSCs-Exo each.The Sham group and the SCI group were given the same volume of PBS.72 hours after the establishment of the model,the spinal cords of each group were collected for TMT quantitative proteomics to find differential proteins and GO and KEGG pathway enrichment analysis.Further analyze the found complement proteins and verify the proteomics data by RT-PCR.Clq immunohistochemical staining and complement-related inflammation NF-κB western blotting were performed.PKH67-labeled BMSCs-Exo combined with immunofluorescence detection of microglia to detect whether BMSCs-Exo was received by spinal cord tissue microglia.Results:Using proteomics,7215 proteins were jointly identified in the Sham group,SCI group and SCI+Exo group.Among the co-identified proteins,comparing the SCI group and the Sham group,the total number of proteins with significant differences was 839,of which 584 were significantly up-regulated and 255 were significantly down-regulated.Among the co-identified proteins,SCI+Exo group and the SCI group,the total number of proteins with significant differences was 121,of which the total number of significantly up-regulated proteins was 10,and the total number of significantly down-regulated proteins was 111.Further annotation and enrichment analysis of these differential proteins found that the complement system is quite different.Comparing the differential proteins of the complement system between the SCI group and the Sham group,24 proteins were significantly up-regulated;comparing the SCI+Exo group with the SCI group,7 significantly down-regulated proteins were found in these 24 complement proteins.RT-PCR further verified this change.Immunohistochemical analysis showed that there were fewer Clq-positive cells in Sham spinal cord tissue specimens,while there were more Clq-positive cells in the SCI group.Compared with the SCI group,BMSCs-Exo treatment reduced the expression of C1q in the SCI+Exo group.The results of immunofluorescence showed that Iba-1 was abundantly expressed near the injured part of the spinal cord tissue of SCI rats.A large number of labeled BMSCs-Exo were found in microglia,most of which were gathered in small clusters in the cytoplasm of microglia.The results of western blot analysis showed that compared with the Sham group,the SCI injury caused a significant increase in phosphorylated NF-κB（p-p65）levels.After exosomes treatment,p-p65 were significantly down-regulated in the SCI+Exo group compared with the SCI group.Conclusion:BMSCs-Exo can inhibit complement mRNA synthesis and release and inhibit activation of NF-κB signaling by binding to microglia cells.Accordingly,BMSCs-Exo may serve as a potential treatment for acute SCI,and the complement system may represent a primary target associated with the beneficial effects of this treatment. |
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