| Objective:By establishing an effective incubation model between the microglial cell(BV2)exosomes and the PD model of PQ-treated MN9 D cells to explore the effect of inflammatory exosomes on the degenerative changes of MN9 D cells and the PD model constructed by MN9 D cells,and hope to reveal the transmission and biological regulation mechanism of functional miRNAs between microglia-exosomes-dopaminergic neurons,which provide a basis for the study of the mechanism of exosomes secreted by microglia on pathological neurons.Methods:1.Extract exosomes in the supernatant of BV2 cells by gradient centrifugation combined with filtration,and observe their morphology with electron microscope;Western Blot detects exosomal marker proteins CD63 and TSG101;uses nanoflow meter to detect the concentration and particle size of exosomes.2.The particle concentration of exosomes released from BV2 cells after 40 μM PQ treatment was detected by a nanoflow cytometer;Western Blot was used to detect the expression level of Rab27 a protein and the expression level of exosomal marker protein CD63 after 40 μM PQ treatment of BV2 cells(n =3);3.Label the exosomes of BV2 cells with PKH26 fluorescent dye,and co-culture them with MN9 D for 6 h,12 h,and 24 h,and observe the interaction between exosomes and MN9 D cells by laser scanning confocal microscopy;label the exosomes After co-cultured with MN9 D cells treated with 100 μM PQ for 24 hours for 12 hours,the uptake of MN9 D cells was observed by laser scanning confocal microscope(n=3);4.Model of effective interaction between BV2 cell exosomes and MN9 D cells(EXO-PD model): After 40 μM PQ treatment of BV2 cells,the exosomes in the supernatant were extracted,and they were pretreated with MN9 D cells for 12 h before PQ treatment After 24 hours,perform subsequent phenotyping experiments on MN9 D cells;5.Different doses of PQ were used to treat MN9 D cells for 24 hours,and the effect of in vitro PD model construction was tested by Parkin/PINK1 protein expression;6.Changes in PD-related phenotypes in the EXO-PD model.The CCK8 method detects the proliferation activity of the EXO-PD model(n=6);Western Blot detects the PD-related proteins(Parkin,PINK1,TH)and apoptosis-related proteins(Bax,Bcl2,caspase 3,caspase 9)of the EXO-PD model Expression(n=3);immunofluorescence method was used to detect the expression of proliferation-related genes ki67 and α-synuclein in the EXO-PD model(n=3);Flow cytometry to detect the apoptotic level of EXO-PD model(n=3).7.High-throughput sequencing to screen the differential miRNAs in the exosomes of BV2 cells after PQ treatment,use miRanda software to analyze the target genes of the differentially expressed miRNAs,and use GO analysis and pathway analysis to screen the target genes;qRT-PCR to verify the expression of differential miRNAs in the intracellular and released exosomes of PQ-treated BV2 cells;qRT-PCR detects the expression of differential miRNAs in MN9 D cells treated with different concentrations of exosomes;8.Use qRT-PCR to detect the expression of differential miRNAs in the EXO-PD model;use small interfering RNA to functionally inhibit miR-92a-3p in BV2 cells and extract exosomes from the supernatant,and qRT-PCR to detect the differential miRNAs Expression;Small interfering RNA is used to functionally inhibit miR-92a-3p and miR-24-3p in BV2 cells and MN9 D cells,and Western Blot detects the protein expression of predicted target genes.Result:1.Under the electron microscope,it was observed that the extracted exosomes sample had a saucer-like specific structure with a double-layer membrane.The nanoflow cytometer showed that the particle size ranged from 50-150 nm.Western Blot detected the marker protein CD63 of exosomes.,TSG101,no negative protein Calnexin was detected,which met the exosomes extraction sample standard.2.Nanoflow cytometry detected that the concentration of particles extracted from the supernatant of microglia treated with paraquat increased,and the expression of CD63 protein was significantly increased.PQ treatment of BV2 caused a decrease in the expression of the vesicle trafficking regulator protein Rab27 a.3.The 3D layer scan of the laser scanning confocal microscope proved that MN9 D cells ingested BV2 exosomes through internalization,and the uptake amount increased with the increase of incubation time,and the uptake amount reached the peak at about 12 h;after PQ treatment of MN9 D cells The content of internalized BV2 cell exosomes decreased significantly.4.Treatment of MN9 D cells with different doses of PQ for 24 hours can cause a significant decrease in the expression of Parkin and PINK1 proteins;the exosomes of BV2 cells increase the proliferation activity of MN9 D cells under normal conditions,and the same exosomes inhibit the proliferation activity of PQ-treated MN9 D cells;5.BV2 cell exosomes caused an increase in the expression of Parkin,PINK1,TH,and p-TH(Ser40)proteins in normal MN9 D cells,and after PQ-treated MN9 D cells ingested exosomes,Parkin,PINK1,TH,and p-TH(Ser40)protein is low expression;PQ treatment increases the expression level of α-synuclein in MN9 D cells,α-synuclein is obviously aggregated in the cell,and PQ-MN9 D cells ingest BV2 cell exosomes,further Increased the expression of α-synuclein;Ctrl-EXO decreased the expression of α-synuclein in normal MN9 D cells;6.After MN9 D cells ingest the exosomes of BV2 cells,the expression of Bax,cleave-caspase 3,cleave-caspase 9 protein increased,and the expression of Bcl2 protein decreased;pro-caspase 3,pro-caspase 9 were cultured and spliced.The protein trend is the same.Incubating the exosomes of BV2 cells with PQ-MN9 D cells caused the expression of Bax,cleave-caspase 3,and cleave-caspase 9 to decrease in MN9 D cells;the expression of Bcl2 increased.7.PQ treatment caused changes in the expression profile of miRNAs in BV2 cell exosomes.A total of 227 differentially expressed miRNAs were screened,and it was found that PQ treatment caused the overall relative expression of total miRNAs in BV2 cell exosomes to be significantly higher than the control group;8.PQ treatment of BV2 cells caused a decrease in the expression level of miR-92a-3p in their cells,a significant increase in miR-24-3p,and an increase in the expression levels of miR-92a-3p and miR-24-3p in their exosomes;1×105/partical number of exosomes can significantly increase the expression of miR-92a-3p in MN9 D cells,while miR-92a-3p in MN9 D cells does not increase significantly when incubated with a higher number of exosomes;MN9D The expression level of cell miR-24-3p does not change significantly;9.Ctrl-EXO caused a slight increase in miR-92a-3p in normal MN9 D cells,and the expression of miR-24-3p remained unchanged,while exosomes caused miR-92a-3p in MN9 D cells in a PQ-damaged state,The expression level of miR-24-3p decreased significantly;after functionally inhibiting the expression of miR-92a-3p and miR-24-3p in BV2,MN9 D cells,the expression of Rap1 b,PTCK1,and Per2 proteins increased;10.The expression of Rap1 b protein was significantly decreased in PQ-treated MN9 D cells,and knocking down miR-92a-3p alleviated the effect of PQ-decreasing Rap1 b.Conclusion1.Paraquat treatment promotes microglial cells to increase the release of exosomes,and inhibits dopaminergic neuronal cells from taking up exosomes from microglial cells.2.Microglial exosomes change the proliferation activity of dopaminergic neurons,the expression level of PD-related proteins and the level of apoptosis.Under normal physiological conditions,they can delay degenerative changes,while in the PD damage model induced by PQ It played a role in exacerbating degenerative changes.3.Physiological and pathological conditions MN9 D cells received microglial exosomes and the expression changes of miR-92a-3p and miR-24-3p were different,indicating that the status of recipient cells also changed the phenomenon of gene delivery.4.miR-92a-3p participates in PQ-induced neurodegenerative damage by inhibiting the expression of target genes per2 and Rap1 b protein. |
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