Preparation And Identification Of Exosomes Secreted By IL-10 Treatmented Immature Dendritic Cells

Chapter One Preparation and identification of exosomes secreted by IL-10 treatmented immature dendritic cellsObjective:To study the effective method for preparation and identification of exosomes secreted by IL-10 treatmented immature dendritic cells(imDC).Methods: Bone marrow progenitor cells were obtained from F344 rat femoral and tibial bones and then cultured in the culture medium with 5ng/ml GM-CSF. On two days, half of the medium was removed and equal volume of fresh medium was added. On day 5, non-adherent and loosely adherent cells were harvested and identified as imDC by flow cytometry, electronic microscope and mixed lymphocyte reaction (MLR). Co-culture with IL-10 for 72h then ollected the cell supernatants using ultracentrifuge method to obtain the exosomes secreted by IL-10 treatmented immature dendritic cells(IL-10-EXO).Results :On day 5 of culture, imDC were harvested and identified. Morphological analysis by electronic microscope showed the typical propery of imDC. Surfacephenotype assay by flow cytometry indicated the low to moderate expression of MHCclassⅡ, and low expression of CD80 and CD86 of imDC. MLR meant that imDC induced lower T-cell proliferation than mature DC. Conclusion: It is an efficient and feasible method in this study of inducing and proliferating rat bone marrow-derived iDC in vitro.The IL-10-EXO had a more significant role in inhibiting inflammation, laid the foundation for further study. Chapter Two Interleukin-10 treated dendritic cells derived exosomes can inhibit trinitrobenzene sulphonic acid induced rat colitisObjective:To investigated the therapeutic action of Interleukin-10-treated dendritic cells derived exosomes for trinitrobenzene sulphonic acid induced rat colitis.Methods:40 male Wistar rats were divided into four groups, n = 10.A group as the control group given an enema of 45% alcohol. Others were given a TNBS enema.3 days after that, according to different treatment methods, 30 rats were divided into three groups: intraperitoneal injection of 1ml saline as B group; intraperitoneal injection of 1ml saline containing 10μg IL-10/EXO as C group; intraperitoneal injection of 1ml saline containing 10μg exosomes as D Group. Observation for 7 days, recorded the clinical activity score of colitis, colon macroscopic scoring system, colon histological scoring system, colon tissue myeloperoxidase(MPO), INF-γ, IL-2, TNF-αand IL - 10 levels. All groups were executed on the 8th day.Results:Compared with B and D groups, C group had a significant lower colitis activity scores, colon scores and colon histopathology scores. Also C group of rats with colitis activity score, colon scores and colon histopathology eye score than those in B, D groups significantly decreased the MPO levels in colon tissue, and promote inflammation silver INF-γ, IL-2, TNF-αin levels are low, suppressing inflammatory cytokines IL-10 levels are higher.Conclusion:Interleukin-10 treated dendritic cells derived exosomes can inhibit trinitrobenzene sulphonic acid induced rat colitis.