The Experimental Study Of The Effect On Immunoregulating Capability Of Exosomes Secreted By MSCs Followed With Different Preconditionings

Objective: To study the effect of the different pretreatments intervene in the culture microenvironment on the immunomodulatory properties of the exosome secreted by mouse bone marrow derived mesenchymal stem cell(MSC),in order to further optimize the MSC derived exosome and make it get better immunomodulatory capability.Methods: The MSCs were divided into four groups based on the different pretreatment means:A, cultured in a normal environment;B,cultured in a hypoxia environment in advance;C,co-cultured with IFN-γ;D, cocultured with both IFN-γ and TNF-α.Then exosomes were extracted from the supernatant of different groups.First,compare the secrection levels of exosomes in these groups,and then observe whether the exosome marked with CM-Di I can be uptaked by the spleen lymphocyte.Based on these results,MSC-exosomes were co-cultured with spleen lymphocytes in vitro to study the effects of exosomes from different groups on the proliferation,differentiation and cytokine secretion of T lymphocytes. An allogeneic delayed type hypersensitivity(DTH) experiment was performed to preliminarily study the exosome function in vivo.Results: The production of exosomes in hypoxia, IFN-γ, IFN-γ + TNF-α pretreatment groups were higher than the normal culture group(P<0.05),in addition,the hypoxia pretreatment group was highest campared with the two others(P<0.05); Compared with the control group,each of the exosome treated group exhibited a obvious inhibition to lymphocyte proliferation,but there was no statistical difference between the pretreatment groups with each other(P>0.05); Compared with the control group,each of the exosome treated group up regulated the expression level of FOXP3 in T lymphocytes and increased the proportion of Treg(P<0.05),and the IFN-γ, IFN-γ + TNF-αpretreatment groups had higher inducibility than the two others(P<0.05); Compared with the control group,each of the exosome treated group promoted T lymphocytes secreted more IL-4, IL-10(P<0.05),especially in the IFN-γ pretreatment group(P<0.01); Each mouse had a lower degree of footpad swelling in exosome treated groups compared with the control group(P<0.05),but there was no obvious statistical difference between the pretreatment groups with each other(P>0.05).Conclusions: MSC prestimulated by hypoxia or inflammatory cytokines could secret more exosomes, especially after the hypoxia preconditioning,the production increased more remarkable. Exosomes secreted by MSC under different preconditions could effectively inhibit the proliferation of T lymphocytes. The exosome derived from MSC pretreatmented with inflammatory cytokines could significantly promote the expression of FOXP3 and induce a Treg expansion, which has a better immunoregulating capability.