Isolation And Identification Of Exosomes Produced By Mouse Bone Marrow Immature Dendritic Cells Transfected With FoxP3 Overexpressing Adenovirus

Objection:FoxP3 overexpression bone marrow-derived immature dendritic cells can induce immune tolerance.The exosomes they produced have lots of advantages and are the potential therapeutic ways to immune disease.In this study,we first cultured mouse bone marrow-derived immature dendritic cells and transfected them with FoxP3 overexpressing adenovirus and isolated and identified the exsomes,then test the FoxP3 the imDCs produced.Method:1 Culture and purify mouse bone marrow-derived imDCsSacrifice the mice and take out their femurs,flushed out bone marrow,lysisd the red blood,and the remaining cells were cultured in a cell culture medium containing fetal bovine serum and cell stimulatory factors.The medium was changed at 48h and 96h respectively,and harvested the cells on the 6th day.Counted the cells,added 100?l of CD11c immunomagnetic beads and 400?l buffer per 10~8 cells,incubated them at 4°C for 15min.Collected the positive cells.2 FoxP3 overexpressed adenovirus transfected imDCsCentrifuged and discarded most of the cell supernatant.According to the transfection complex MOI=500,the corresponding virus was added and cultured for 15 minutes.Then cells were plated in 24-well plates at a cell concentration of approximately 10~5/ml and cultured for 24h.Cells were harvested,washed,and cultured for 96h with RPMI-1640 containing exogenous fetal calf serum.3 Identification of FoxP3 expression secreted by imDCsFluorescence microscopy observe of FoxP3 expression during cell culture.At the 72h,collect the cells and centrifuged.Western-blot to detect the expression of FoxP3.4 Separation and purification of imDexCollect cell supernatant and centrifuged at 10000 rpm for 30 minutes to remove cell debris.Add the exosome extraction kit,mixed and keep it at room temperature for 12 hours.Centrifuged and collect exsome.5 Identification of exosomes and analysis of exosomal FoxP3 proteinObserved exsome by transmission electron microscope.Detection of Dex-specific marker CD63 by Western-blot.6 Western-blot detects imDex FoxP3 expression.Results:1 Observation of imDC by morphologyOn the 48h after seeded,the cells appeared round and scattered.The 96h:The cells appear fine protrusions and colonies grow.Day 6:The cells showed dendritic protrusions and the colonies spread apart,consistent with the immDC growth pattern.After immunomagnetic beads sorting,high magnification under light microscope showed that the cell viability was good,and most cells had irregular dendritic processes.2 Identification of imDC FoxP3 after transfectionFluorescence microscopy observation:At 12h after transfection,the cells showed good viability and appeared fluorescence.At the 96h,the cell showed high fluorescence and the viability became worse,some of the cells died.96h after transfection,Western-blot showed that the expression of FoxP3 in the FoxP3 overexpressing adenovirus group was significantly higher than that of the control group(the same titer as the blank adenovirus)(P<0.05).3 Mouse bone marrow imDex identificationTransmission electron microscopy observed that the exosomes were round or oval,slightly concave on one side,and the diameter was about50-100nm,which was in line with the morphological characteristics of exosomes.Western-blot see high expression of the Dex specific marker protein CD63,combined with morphological results,to demonstrate that the extracts were exosomes.4 imDex FoxP3 testThe exsome of FoxP3 overexpressing adenovirus group was significantly higher than that of the control group(the same titer as the blank adenovirus)(P<0.05)Conclusion:1 We successfully used FoxP3 overexpressing adenovirus to transfect mouse bone marrow-derived imDC,and we detected high expression of FoxP3 in imDCs.2 We purified exosome the imDC secreted and observed exosome by electron microscopy and detected exosome-specific markers CD63,which strongly demonstrated that the extracts were exosomes.3 We successfully detected high expression of FoxP3 in exosomes produced by FoxP3 overexpressing adenovirus group,demonstrating that they carry imDC immunoregulatory information.4 This experiment innovatively uses imDex as small molecules to penetrate the blood-brain barrier and provides the experimental basis for the treatment of MS and other neuroimmunological diseases.