Glioma Cell-derived Exosomes Rich In MiR-124-3p Inhibit Glial Scar Formation And Its Mechanism

Astrocytes are the most numerous glial cells in the central nervous system,and their number is more than five times that of neurons.They not only support neurons,but also play an important role in synapse formation,ion homeostasis,neurotransmitter clearance,and regulation of extracellular space volume.Under normal physiological conditions,astrocytes support and protect neurons,maintain ion balance around neurons,regulate neurotransmitter metabolism,and participate in the transmission of synaptic signals;after the central nervous system is injured,the astrocytes around the injured area are activated and transformed into reactive astrocytes.They proliferate and migrate to the injured site.Together with oligodendrocytes and microglia,it forms a glial scar,secretes axon growth inhibitors and prevents axon regeneration,which is one of the main reasons why the regeneration capacity of the central nervous system is limited.Therefore,how to inhibit and eliminate the glial scars formed by reactive astrocytes is the key to the treatment of central nervous system damage.miR-124 is one of the most abundant miRNAs in the central nervous system.It plays an important role in many physiological and pathological processes by regulating autophagy,neuroinflammation,oxidative stress,neural differentiation,and mitochondrial function.Studies have shown that miR-124 reduces the formation of glial scars by targeting STAT3 and helps restore nerve function.Some researchers have found that astrocytes differentiate into neurons after overexpression of miR-124,thereby inhibiting activation.These studies show that miR-124 can not only inhibit the formation of glial scars,but also benefit the reconstruction of neural networks and the recovery of nerve tissue functions.Exosomes are vesicles secreted by cells,with a typical lipid bilayer membrane,wrapped with proteins,lipids and various nucleic acids(m RNAs,miRNAs,lnc RNAs).Exosomes are a natural intercellular signal transmission carrier,which can efficiently deliver miR-124 and other biologically active substances,thereby regulating cells and playing an important role in the treatment of central nervous system damage.In order to obtain a large number of exosomes carrying biologically active substances,we need to select appropriate donor cells.Glioma cells are the most common intracranial primary malignant tumor cells.There are many commercial cell lines,and they may be excellent donors for the preparation of large amounts of exosomes carrying biologically active substances and used for central nervous system damage.Therefore,this experiment plans to use the commercialized U-87 MG human glioma cell line(U-87 cells)as a donor to prepare miR-124-3p-rich exosomes.Explore the role and mechanism of this kind of exosomes in the formation of glial scars,and evaluate its safety,conduct innovative basic research and provide basic data and theoretical basis for the treatment of central nervous system damage with exosomes of glioma cells.This study is mainly divided into the following contents:(1)Extract primary astrocytes from the brain of r rats,use immunofluorescence to identify the cell characteristics and purity,stimulate it with TGF-β1 to activate and become reactive astrocytes;(2)Choose commercial U-87 cells as the donor of exosomes,separate the exosomes from the supernatant by ultracentrifugation,observe and measure the shape and size of the exosomes with a scanning electron microscope,and use Western blot analyze the expression of exosomes markers CD9,CD63 and TSG101,analyze the size distribution of exosomes with NTA,stain the exosomes with PKH67,and observe whether reactive astrocytes absorb exosomes under a fluorescence microscope;(3)Two methods were used to construct exosomes overexpressing miR-124-3p,the expression of miR-124-3p was detected by RT-q PCR,the exosomes and reactive astrocytes were cultured together,RT-q PCR was used to detect the changes in the content of miR-124-3p in the cells,Western blot and RT-q PCR were used to detect the expression of GFAP and SOX9 related to activation and STAT3 and p-STAT3 related to scar formation,the pros and cons of the two methods are discussed;(4)miR-124-3p mimic labeled with cy3 fluorescein and modified with cholesterol and exosomes are incubated together at 37°C to prepare exosomes rich in miR-124-3p,And then process reactive astrocytes,observe them with an inverted phase contrast microscope;(5)Use EdU and miR-124-3p-rich exosomes to treat reactive astrocytes together,and use immunofluorescence to analyze cell proliferation;(6)Use high-throughput analysis of the differentially expressed m RNA in the reactive astrocytes treated with exosomes,and infer the mechanism of action of the exosomes;(7)Transplant exosomes into rats with spinal cord injury,detect the formation of glial scars and observe whether tumors appear after transplantation.The results are as follows:(1)Astrocytes were successfully extracted from the brains of neonatal rats,and immunofluorescence was used to identify the astrocyte marker GFAP as a strong positive expression;after further purification with cytarabine,the cell purity reached more than 95%;(2)U-87 cell exosomes were obtained by ultracentrifugation.A disc-like structure with a diameter of about 100 nm was observed under the electron microscope;the expression of the exosomes markers CD9,CD63 and TSG101 was detected by Western blot;The particle distribution index(PDI)obtained by NTA analysis is between 0.08-0.7,and the substance with a diameter of 30nm-150 nm accounts for 56%;PKH67-labeled exosomes(green)were observed with a fluorescence microscope to be located in the cytoplasm of reactive astrocytes,indicating that the exosomes were absorbed;Method 1: Transfect miR-124-3p first,and then isolate exosomes.(3)U-87 cells were transfected with 50,100,200 n M miR-124-3p mimic,and RT-q PCR detected that the miR-124-3p content in the cells increased by about 4,6,and 6 times,but compared to other concentrations,when 200 n M is used,the number of U-87 cells is significantly reduced,so 100 n M is chosen as the optimal transfection concentration.The U-87 cell supernatant was collected,and the exosomes were separated by ultracentrifugation.The miR-124-3p in the exosomes was detected by RT-q PCR,which increased by 4 times.The exosomes overexpressing miR-124-3p were added to the reactive astrocyte culture medium at a concentration of 70 μg/ml,and after 48 hours of incubation,RT-q PCR detected that the expression of miR-124 in reactive astrocytes increased by 1.8 times,and Western blot was used to detect that GFAP and SOX9 were down-regulated in protein levels,and the phosphorylation level of STAT3 decreased.Method 2: Isolate exosomes first,and then increase the expression level of miR-124-3p by co-incubation.The exosomes were collected from U-87 cell culture medium by ultracentrifugation,incubated with cholesterol-modified miR-124-3p mimic at 37°C for 1 h,washed with PBS buffer,centrifuged again,and obtained exosomes.The content of miR-124-3p in exosomes increased 15 times.The exosomes overexpressing miR-124-3p were added to the reactive astrocyte culture medium at a concentration of70μg/ml and co-cultured for 48 h.RT-q PCR detected an 8-fold increase in the expression of miR-124-3p in reactive astrocytes,activation-related GFAP,SOX9 and scar-related STAT3 were significantly down-regulated at the gene level.It was detected by Western blot that GFAP and SOX9 were down-regulated at the protein level,and the phosphorylation level of STAT3 decreased.Compared with the first method,the decrease was more significant.In summary,the operation of the second method is simple and convenient,the improvement of miR-124-3p is more significant,and it effectively inhibits the phosphorylation of STAT3 and the expression of reactive glial cell markers such as GFAP and SOX9.This is the best strategy for constructing exosomes overexpressing miR-124-3p in vitro.Follow-up experiments use method two to prepare exosomes rich in miR-124-3p.(1)Prepare cholesterol-modified cy3-labeled exosomes rich in miR-124-3p,and process reactive astrocytes.Compared with the control group,the experimental group contains a large amount of cy3 fluorescent substances,which proves reactive astrocytes take up miR-124-3p in large quantities by ingesting exosomes;(2)The EdU experiment proved that exosomes overexpressing miR-124-3p can inhibit the proliferation of reactive astrocytes(P<0.05);(3)Exosomes overexpressing miR-124-3p regulate multiple genes(Gfap,Vim,Cspg5,Sox9,S100 b,Cdk1)and pathways(c AMP,Jak-STAT,m TOR,PI3K-Akt,TGF-beta),it is speculated that it may use multiple pathways to coordinately regulate the scar formation process after injury;(4)Exosomes overexpressing miR-124-3p can be taken up by astrocytes in vivo and inhibit their proliferation,helping to reduce glial scar formation.Conclusion:(1)The co-incubation of cholesterol-modified miR-124-3p mimic with exosomes can effectively prepare miR-124-3p-rich exosomes.Compared with the traditional method of transfection and then isolation,the method is more efficient and the operation is more convenient;(2)Exosomes rich in miR-124-3p can be absorbed by reactive astrocytes,down-regulating multiple genes and pathways related to growth and proliferation,extracellular matrix secretion,and glial scar formation in reactive astrocytes,effectively inhibit the activation of astrocytes and the formation of glial scars.