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The Mechanism Of CD73+hucMSC-EVs Ameliorate Spinal Cord Injury By Promoting Polarization Of M2 Microglia

Posted on:2023-05-09Degree:MasterType:Thesis
Country:ChinaCandidate:H YangFull Text:PDF
GTID:2544306614481944Subject:Surgery (bone)
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Spinal cord injury(SCI)is a life-threatening and devastating injury that causes temporary or permanent changes in the spinal cord with partial or complete loss of motor,sensory,and autonomic nerve function,with sensory dysfunction generally below the level of injury.It is estimated that the prevalence and incidence of SCI in the world is 236-4187/1million,with as many as 770,000 new cases per year,mostly in men under 30 years old.Because severe cases can lead to paralysis,usually paraplegia or quadriplegia,it can place a serious psychological and financial burden on patients and the healthcare system.After years of research,although the mechanism of spinal cord injury has a certain understanding,the existing treatment methods mainly include drug therapy,surgical decompression surgery and cell therapy,but none of them can achieve satisfactory results.With the deepening of basic experiments,we found that human umbilical cord mesenchymal stem cells(huc MSC)have the advantages of multi-differentiation,short proliferation time,easy extraction and long survival time after transplantation.The seed cells are considered to be favorable for transplantation of stem cells for cell therapy,but the limited rate of their transplantation to the damaged core area and the possible tumorigenicity of stem cells further limit their clinical application.Current research results show that transplanted stem cells mainly play a role through paracrine function,and extracellular vesicles(EVs)has become the most valuable paracrine molecule for its special double-layer lipid membrane structure that can easily cross the blood-brain barrier.When acute injury occurs,macrophages form the first line of defense and can migrate to the site of injury,secreting inflammatory cytokines and engulfing foreign bodies.After spinal cord injury,infiltrating macrophages and microglia in situ generally reach their peak3-7 days later,and rapidly differentiate into M1 subtype with pro-inflammatory effect,which leads to secondary injury with further secretion of pro-inflammatory cytokines.On the other hand,macrophages and microglia can also naturally polarize into the anti-inflammatory M2 phenotype,thereby reducing the inflammatory response.Along with the persistence of inflammation after injury,another type of cell,astrocytes,may be triggered by M1-type microglia,leading to proliferation and cell scarring of A1 phenotype astrocytes,which is harmful to the surrounding environment,but generally peaks at about 2 weeks.Therefore,the balance between M1 and M2 subtypes may be a potential target for SCI drug therapy.Studies have shown that in the inflammatory state of spinal cord injury,due to the activation of inflammatory factors,excess adenosine triphosphate(ATP)is released from the cytoplasm to the extracellular space,further amplifying the inflammatory immune response by activating immune cells.Extracellular ATP can be rapidly broken down into adenosine monophosphate(AMP)in response to CD39,but the process of AMP conversion is restricted by CD73(ecto-5 ’-nucleotidase).CD73 acts as a rate-limiting enzyme for extracellular AMP hydrolysis to adenosine.An extracellular nucleotide enzyme that attaches to the plasma membrane and may affect purine nucleotide metabolism.However,as a macromolecule,it is difficult to cross the blood brain barrier(BBB)and has the possibility of tumorogen.However,its effect on SCI is still unknown.The objective of this study was to construct CD73 modified human umbilical cord mesenchymal stem cells-derived extracellular vesicles,namely CD73+huc MSC-EVs and further investigate the anti-inflammatory effects and possible mechanisms of vesicles after spinal cord injury.Part I: Extraction and identification of extracellular vesicles derived from human umbilical cord mesenchymal stem cells with CD73 overexpression and animail experimental studyObjective:1.CD73 sequence was synthesized by gene,and plasmid was introduced to screen stable CD73 overexpressed strains,and extracellular vesicles were classified and collected and identified2.The efficacy of CD73+ huc MSC-EVs was proved by animal experimentsMethods:1.The morphology of CD73+huc MSC-EVs was observed by transmission electron microscope,and its radius was calculated by NTA.2.Western blot assay was used to identify CD73+huc MSC-EVs.3.The SCI model of mice was constructed and the behavior of mice was analyzed by BMS and BBB score.4.In vivo imaging technique was used to trace Di R labeled CD73+huc MSC-EVs,and the fluorescence distribution of mice after SCI was observed to understand its pathway.Results:1.CD73+huc MSC-EVs showed double concave disk.NTA results showed that the diameter of CD73+ huc MSC-EVs was about 100nm;2.Western blot results showed that CD73+huc MSC-EVs expressed specific proteins :CD73,CD63,CD81;3.In animal behavior analysis,compared with model group from day 7,BMS and BBB scores of CD73+huc MSC-EVs group were higher,P <0.05;4.In vivo imaging system(IVIS)results showed that CD73+huc MSC-EVs did not fluoresce in the original site of T8-T9 spinal cord injury,and the fluorescence was mainly distributed in the abdominal cavity and distributed in the kidney and liver,proving that CD73+huc MSC-EVs did play a role and was metabolized by kidney and liver;Conclusion: We established CD73+huc MSC-EVs drug vector by engineering EVs and demonstrated its efficacy in SCI mice.Part II: Extracellular vesicles derived from human umbilical cord mesenchymal stem cells with CD73 overexpression ameliorate spinal cord injury by activating adenosine receptor A2 b and c AMP/PKA signaling pathwayObjective:1.Whether CD73+huc MSC-EVs can promote the polarization of M2/M1 microglia and thus exert the anti-inflammatory effect of M2 type;2.What are the possible receptors and signaling pathways that CD73+huc MSC-EVs plays a role in.Methods:1.Death and survival of spinal cord cells were analyzed by H&E,Tunel and Nissl staining;2.Immunofluorescence labeled Nitric oxide synthase(i NOS)and Arginase-1(Arg-1)as surface markers of M1 and M2 cells,respectively,to observe the polarization of M1/M2 microglia in the treatment of CD73+huc MSC-EVs;3.A2 a R and A2 b R inhibitors knock out cells to investigate adenosine receptor activation;Microglia biomarkers and c AMP/PKA levels were detected;4.Repeated in vivo studies,morphological staining,flow cytometry,cytokine analysis,and ELISA were used for validation.Results:1.H&E staining,Tunel staining and Nissl staining showed that compared with SCI group,the nerve mortality was lower in CD73+ huc MSC-EVs group,and the morphology of spinal cord tissue cells was improved(P <0.05);2.Immunofluorescence of i NOS and Arg-1 on the surface of M1 showed that M2-labeled Arg-1 was significantly increased in CD73+huc MSC-EVs group,while M1-labeled i NOS was significantly decreased(P<0.05),which promoted the polarization of M2/M1microglia;3.MRS1706(A2b R inhibitor)was significantly decreased,while SCH58261(A2a R inhibitor)was not significantly decreased when treated with CD73+huc MSC-EVs.At the same time,c AMP and PKA were decreased in the short hairpin RNA adenosine A2 b receptor(sh RNA A2 b R)group and CD73+huc MSC-EVs group,and the level of Arg-1 in the sh RNA A2 b R group was significantly lower than that in the sh RNA scrambled group.The i NOS level in sh RNA A2 b R group was significantly increased.At the same time,the expression markers of M1 m RNA(TNF-α,IL-1β and CD86)were increased,while the expression markers of M2 m RNA(Arg-1,IL-10 and CD206)were decreased;4.Flow cytometry results showed that M2:M1 was calculated by CD206 and CD86 biomarkers of M2 and M1 in SCI+CD73+ huc MSC-EVs group,and the ratio of M2:M1 was significantly higher than that in other groups.Cytokine analysis: In SCI+CD73+huc MSCEVs group,pro-inflammatory cytokines IL-1β,IL-6,TNF-α,MCP-1,IFN-α and MIP-1βwere significantly decreased,while anti-inflammatory cytokines such as IL-4 and IL-10 were significantly increased.The levels of TNF-α,IL-6 and IL-1β in SCI+CD73+ huc MSCEVs group were increased(P <0.05),and the levels of IL-10,an important anti-inflammatory cytokine,were significantly decreased in SCI+CD73+ huc MSC-EVs group.Conclusions: CD73+huc MSC-EVs promotes the hydroxylation of ATP to adenosine.CD73+huc MSC-EVs can improve the inflammatory response after spinal cord injury and regulate macrophages/microglia M2:M1 by activating A2 b R/c AMP/PKA signaling pathway.
Keywords/Search Tags:CD73, extracellular vesicles, human cord mesenchymal stem cells, spinal cord injury, M2/M1 microglia
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