Investigation Of Mesenchymal Stem Cells Derived Exosomes In Corneal Allograft Rejection

Objective Corneal transplantation enjoys a lower rejection rate than other organ thansplantations.Immunological rejection postoperatively is considered to be the first cause of surgery failure.It has been thought that MSCs play a role in a paracrine way and MSCs derived exosomes seems to be the functional undertaker.Considered of the promising effect of MSCs on immun rejection,we futher tested the immunomodulate effect of MSCs derived exosomes on a rat corneal allograft rejection model.Methods 1.Flow cytometry was used to test the phenotype of cultured MSCs.MSCs were cultured in conditional media for differentiateon inducing test.2.MSCs-exo was obtained and has typical exosomes sutucture and charactered by protein marker CD9,CD63 and CD81.3.Set up cornea allograft rejection model.Wistar rats were used as donors,while Lewis rats were used as recipients.Transplanted rats were treated into different groups randomly: Group A(control group,model group);Group B(MSCs subconjuctival injection group)Group C(PBS subconjuctival postoperatively injection group);Group D: MSCs-exo postoperatively subconjuctival injection groups Group Da:(1μg MSCs-exo treated group);Group Db:(10 μg MSCs-exo treated group);Group Dc:(100 μg MSCs-exo treated group);Group E(PBS subconjuctival injection preoperatively);Group F(10μg MSCs-exo subconjuctival injection group preoperatively);Group G(PBS subconjuctival injection when rejection happened);Group H(10μg MSCs-exo subconjuctival injection when rejection happend);Group I(PBS was treated as eye drops);Group J(MSCs-exo was treated as eye drops);Group K(PBS infusion postoperatively via tail vein);Group L(MSCs infusion postoperatively via tail vein).Grafts were under observation after surgery.Value the degrees of transparency,edema and extent of neovascularization and compared survival time of each group.4.Collect mononuclear cells from spleen and lymph nodes of Group C and Db,and studied T-cell proliferation by a standard 3H-thymidine incorporation assay.5.ELISA kit was used to measure the levels of Th1,Th2 and Th17 cytokine from MNCs cultured supernatants and cornea tissure.6.Evaluate the expression of CD4,CD25 and Foxp3 form spleen anad lymph nodes of Group C and Group Db by flow cytometry.7.Four corneas from same group were treated as one sample.Gene chip technique was used to valuve the change of m RNA expressing in Group C and Group Db.Results 1.MSCs were obtained and verified for its differentiation ability.Red adipose vacuole can be observed in adipose-like cell and blank calcium salts deposition was found in bone-like cells.2.MSCs-exo was obtained and has typical exosomes sutucture and charactered by protein marker CD9,CD63 and CD81.3.Set up Wistar/Lewis penetrating keratoplasty(PKP)model.The rejection time was around 10 days.4.Subconjuctival injection is much more effective among the three treated routes.5.The dose of 10μg injection of MSCs-exo leads to longer suvival time of corneal allografts.MSCs-exo nfused postoperation 6.Post-op injection of MSCs-exo turns out to have a promising result in allograft survival.7.T cells proliferation was inhabited in MSCs-exo treated group.8.The ratio of IL-10/IFN-γ was upgraded while IL-17 level was decread in cornea.The proportion of CD4+CD25+Foxp3+ Tregs was also increase in MSCs-exo treated group.Conclusions MSCs-exo can effectively inhabit immunological rejection,through the restrain of pathogenic T-cell responses,modulate CD4+T cells differiation and increasing the proportion of regulatory T cells.Th1 pathway is obviously inhabited in the study.JAK-STAT pathway and MAPK pathway were involved in MSCs-exo treatment process.