| Background:Primary Sjogren’s syndrome(p SS)is a chronic autoimmune disease characterized by lymphocyte proliferation and progressive injury of exocrine-glands.At present,symptomatic treatment is the main treatment.When systemic lesions occur,hormones and anti-rheumatic drugs are used to improve the condition and control active inflammation,but satisfactory therapeutic effects have not been obtained.Therefore,it is urgent to find a safe and effective treatment strategy.The etiology of p SS is not clear,and may be related to genetic,environmental,immune and other factors.Studies have shown that CD4~+T lymphocytes play a key role in the early onset of p SS.Mesenchymal stem cells(MSCs),as a kind of pluripotent stem cells,have been widely used in many studies of autoimmune diseases,hematological diseases,cardiovascular diseases,etc.,but as cell therapy,MSCs have limitations such as capillary blockage,tumorigenesis and ectopic osteogenesis.Recent studies have found that the immune regulation and tissue repair ability of MSCs are mainly due to its paracrine effect.The mesenchymal stem cell exosomes(MSC-Exos)derived from labial glands can inhibit the differentiation of Th17cells,increase the salivary flow rate,improve the dryness like symptoms,reduce the level of autoantibodies,and reduce lymphocyte infiltration foci in mice with Sjogren’s syndrome.Compared with other sources of MSC-Exos,umbilical cord derived mesenchymal stem cell exosomes(UCMSC-Exos)have a wide range of sources,convenient materials and no moral and ethical restrictions.However,there is currently no study on UCMSC-Exos in SS,so whether UCMSC-Exos can improve dryness like symptoms in NOD mice,and what effect does UCMSC-Exos have on CD4~+T cells,a key link in the early onset of SS.Objective:1.To study the effects of UCMSC-Exos on SS-Like symptoms in NOD mice.2.To investigate the immunoregulatory effects of UCMSC-Exos on CD4~+T cells in NOD mice.Methods:1.Experimental grouping and intervention plan:NOD model mice and C57BL/6mice were purchased,with C57BL/6 mice as normal control group,NOD-PBS group as blank control group,NOD-UCMSCs group and NOD-Exos group as intervention group,administered once at 8 weeks and once at 9 weeks,respectively(Administration status of each group:C57BL/6-PBS group 100μL PBS,NOD-PBS group 100μL PBS,NOD-UCMSCs group 1×10~6/100μL UCMSCs,NOD-Exos group 100μg/100μL UCMSC-Exos),were sacrificed at 12 weeks.Peripheral blood,spleen and submandibular gland were collected.2.Effects of UCMSC-Exos on SS-Like symptoms in NOD mice:The general condition(body weight and salivary flow rate)of mice was assessed.Peripheral blood of mice was collected and the levels of immunoglobulin(Ig A,Ig G,Ig M)and autoantibodies(anti-SSA antibody,anti-SSB antibody)were detected by ELISA.The submandibular gland of mice was collected and the lymphocyte infiltration was observed by HE staining.3.The regulatory effect of UCMSC-Exos on CD4~+T differentiation in NOD mice:Immunohistochemical detection of CD4~+T cell differentiation in submandibular gland and spleen.4.Effects of UCMSC-Exos on CD4~+T Cell-related inflammatory factors in NOD mice:The peripheral blood of mice was collected,and the levels of CD4~+T-cell associated inflammatory factors(IFN-γ,TNF-α,IL-2,IL-4,IL-6,IL-17A,IL-17F,IL-22,IL-10 and TGF-β)were detected by ELISA.Results:1.Effects of UCMSC-Exos on SS-Like symptoms in NOD mice:(1)General status:Compared with NOD-PBS group,the body weight of mice in NOD-UCMSCs and NOD-Exos groups were increased,but there was no significant between them.Compared with NOD-PBS group,the salivary flow rate of mice in NOD-UCMSCs group showed an increasing trend,but there was no statistical difference.Compared with NOD-PBS group,salivary flow rate of mice in NOD-Exos group was increased.No significant difference was found between NOD-UCMSCs and NOD-Exos group.(2)Compared with C57BL/6-PBS group,Ig A and Ig G levels in NOD-PBS group were significantly increased,and the differences were statistically significant.There was no significant difference in Ig A level between NOD-PBS group,NOD-UCMSCs group and NOD-Exos group.Compared with NOD-PBS group,Ig G in NOD-UCMSCs group and NOD-Exos group was significantly decreased,and the difference was statistically significant.Compared with C57BL/6-PBS group,NOD-PBS group,NOD-UCMSCs group and NOD-Exos group,there were no significant differences in Ig M levels among all groups.Compared with the C57BL/6-PBS group,the serum anti-SSA antibody level of mice in the NOD-PBS group was higher,and the difference was statistically significant.Compared with the NOD-PBS group,the serum anti-SSA antibody of mice in the NOD-UCMSCs group and the NOD-Exos group was significantly decreased,and the difference was statistically significant.Compared with the C57BL/6-PBS group,NOD-PBS group,NOD-UCMSCs group and NOD-Exos group,there was no significant difference in serum anti-SSB antibody levels among all groups.(3)Compared with NOD-PBS group,NOD-UCMSCs group showed a decreased trend of lymphocyte infiltration in submandibular gland,but there was no statistical difference.Compared with NOD-PBS group,NOD-Exos group had significantly less lymphocyte infiltration foci in submandibular gland.There was no significant difference between NOD-UCMSCs group and NOD-Exos group in lymphocyte infiltration foci of mouse submandibular gland.2.Immunoregulatory effects of UCMSC-Exos on CD4~+T cells in NOD mice:(1)Compared with NOD-PBS group,the proportion of IFN-γand IL-17A positive cells decreased in NOD-UCMSCs and NOD-Exos groups,while the proportion of IL-4and TGF-βpositive cells increased.There was no significant difference in the proportion of IFN-γ,IL-4,IL-17A and TGF-βpositive cells between NOD-UCMSCs group and NOD-Exos group.Compared with C57BL/6-PBS group,the proportion of IFN-γand IL-17A positive cells in the spleen of mice in NOD-PBS group was increased,while the proportion of IL-4 and TGF-βpositive cells was decreased.Compared with NOD-PBS group,the proportion of IFN-γand IL-17A positive cells in the spleen of mice in NOD-UCMSCs and NOD-Exos groups decreased,while the proportion of IL-4 and TGF-βpositive cells increased.There was no significant difference in the proportion of IFN-γ,IL-4,IL-17A and TGF-βpositive cells in mouse spleen between NOD-UCMSCs group and NOD-Exos group.(2)Compared with C57BL/6-PBS group,the expression levels of IFN-γ,TNF-α,IL-2,IL-4,IL-6,IL-17A,IL-17F and IL-22 were increased in NOD-PBS group,while the expression levels of IL-10 and TGF-βwere decreased.Compared with NOD-PBS group,the expression levels of IFN-γ,TNF-α,IL-6,IL-17A,IL-17F and IL-22 in NOD-UCMSCs group were decreased,while the expression levels of IL-10 and TGF-βwere increased,while the expression levels of IL-2 and IL-4 were not significantly different.Compared with NOD-PBS group,the expression levels of IFN-γ,TNF-α,IL-2,IL-6,IL-17A,IL-17F and IL-22 in NOD-Exos group were decreased,while the expression levels of IL-10 and TGF-βwere increased,while the expression levels of IL-4were not significantly different.The expression levels of IFN-γ,TNF-α,IL-2,IL-4,IL-6,IL-17A,IL-17F,IL-22,IL-10 and TGF-βin NOD-UCMSCs group were not significantly different from those in NOD-Exos group.Conclusion:1.UCMSC-Exos can increase body weight and salivary flow rate,improve dry symptoms,decrease Ig G and anti-SSA antibody levels,and decrease submandibular gland lymphocyte infiltration foci in NOD mice.2.UCMSC-Exos can regulate T cell differentiation,inhibit the secretion of pro-inflammatory cytokines associated with CD4~+T cells,and promote the secretion of anti-inflammatory cytokines. |
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