Effect Of MSCs Exosomes Activated By TLRs Signaling Pathway On The Polarization Of Macrophages And Study On Isolation Method Of Exosome Microfluidic Chip

BackgroundMacrophages play an important role in the repair of tissue damage.According to their immunoregulatory functions,they can be divided into proinflammatory M1 type and anti-inflammatory M2 type.Under certain conditions,phenotypic transformation can take place,and the polarization direction of macrophages affects the process of tissue repair to a certain extent.It has been found that vesicle system and exosome are also important ways to regulate macrophage polarization.Mesenchymal stem cell exosomes have attracted increasing attention due to their similar biological characteristics to mesenchymal stem cells.Recent studies have shown that activation of different Toll-like receptors on MSCs results in different inflammatory regulators in MSCs,which regulate inflammation through exosome-mediated cytokine transfer.However,the effects of exosomes stimulated by different TLRs signals on macrophage polarization and their roles have not been reported.Therefore,a clear understanding of the interaction between MSCs and macrophages is conducive to a deeper understanding of the potential impact of MSCs on the course of disease in the repair of tissue damage,thus providing new ideas for the clinical application of MSCs-Exo based acellular immunotherapy.Exosomes are currently a research hotspot,they are considered as important biomarkers in clinical diagnosis and treatment.They can reveal the biological information of the source cells and provide a new method for the diagnosis and treatment of clinical diseases.However,the isolation of exosomes is a difficult problem at present.In view of this,it is particularly important to develop an efficient and high purity isolation method for exosomes.In the second chapter,we designed a set of exosome isolation device by combining microfluidic technology with nanopore membrane filtration technology.It has been proved that this method can extract exosomes from biological samples efficiently,which will provide technical support for the study of clinical diseases of exosomes.ObjectiveThis study aims to investigate the characteristics of exosomes derived from bone marrow mesenchymal stem cells,the effects of MSCs activated by different TLRs signaling pathways on macrophage polarization,and the isolation technology of exosomes.Methods1.The primary MSCs were isolated and obtained by whole bone marrow method.The differentiation ability of MSCs was evaluated by cell morphology observation,flow cytometry,surface markers CD90,CD45,CD34,CD44,CD29 and osteogenic adipogenesis induction test,and the characteristics of MSCs were determined.The exosomes were extracted from the supernatants of BM-MSCs activated by different TLRs signaling pathways in the 4-6 generations.The morphology of the exosomes was observed by transmission electron microscopy(TEM),and the surface specific markers CD9 and HSP70 were detected by Western blotting to confirm the characteristics of the exosomes derived from stem cells.2.MSCs-Exo activated by TLRs signaling pathway stimulates macrophages: M0 macrophages were cultured with RPMI-1640 containing 10% FBS and 10% L929 conditioned medium.When the cell density reached 70%-80%,the cells were divided into 6 groups.M0 group(without any treatment),M1 induction group(with LPS 100 ng/mL),M2 induction group(with IL-4 20 ng/mL + IL-13 20 ng/mL),MSCs-Exo group(with MSCs-Exo 20 ?g/mL),MSCs-Exo group(with MSCs-Exo 20 ?g/mL)MSCs-Exo group activated by TLR3(with Poly(i : c)pretreated MSCs-Exo 20 ?g/mL)and MSCs-Exo group activated by TLR4(with LPS pretreated MSCs-Exo 20 ?g/mL),After cultured for 48 h,morphology of macrophages was observed under an electronic microscope.The immunophenotype(CD206,Arg-1,TNF-? and iNOS)and difference of expression of inflammatory factors(CCL22,IL-1?,IL-6 and IL-10)of macrophages in each group were detected by flow and qPCR,respectively.3.Based on microfluidic technology and nanopore membrane filtration technology,a pressure-driven exosome isolation chip was designed.The chip can collect 300 ?L exosome samples after passing 5-10 mL samples through 30 nm nanopore membrane filtration.Results1.BM-MSCs have high self-renewal and osteogenic and adipogenic differentiation ability.They also express the surface markers CD90,CD29 and CD44 of mesenchymal stem cells,but not CD34 and CD45,which conform to the recognized MSCs phenotype.2.The exosomes secreted by BM-MSCs were observed under transmission electron microscopy as round or elliptical bilayer membrane structures with diameters ranging from 40 to 200 nm,expressing surface markers CD9 and HSP70.3.The morphology of macrophages in each group was observed under an electronic microscope,Morphology of long carboxylic shape and many pseudopods were observed in groups withmacrophages stimulated by MSCs-Exo,MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways.High expression of CD206 and Arg-1,and low expression of TNF-? were respectively detected by flow cytometry in the groups with the macrophages activated by MSCs-Exo,MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways,and low expression of iNOS with the macrophages activated by MSCs-Exo,and invariant expression of iNOS with the macrophages activated by MSCs-Exo activated by TLR4 signaling pathways and MSCs-Exo activated by TLR3 signaling pathways.High expression of CCL22 and IL-10,low expression of IL-1? and IL-6 were detected by qPCR.4.The microarray device for extracting and separating exosomes can successfully isolate 300?L exosome biological samples,and the double-layer membrane vesicles with circular or elliptic morphology are observed under electron microscopy.Nanosights identified that the particle size distribution is mostly 30-200 nm.Western blot identified that it expresses specific exosome protein markers HSP70,CD9 and CD81.Conclusion1.BM-MSCs have the characteristics of stem cells and can secrete exosome from MSCs.2.Polarization of macrophages to M2 type can be stimulated by MSCs-Exo activated by TLR3 and TLR4 signaling pathway.3.Microfluidic chip exosome separation device can efficiently separate exosomes from biological samples.