ObjectiveTo explore the influence of miRNA-155/PU.1 signaling pathway blockade on bone marrow-derived dendritic cells (DCs) maturation and immune function of rat small intestinal transplantation. MethodsThe DCs were induced by adherent culture.The critical transcription factor gene PU.1 was designed and PU.1 siRNA was synthe-sized.The DCs were transfected by liposome transfection and a pair of PU.1 siRNA was screened according to the high silencing efficiency.The expressions of DCs surface markers CD80, CD86, and MHC-Ⅱamong three groups (PU.1 silent group, negative control group, and control group) were analyzed by flow cytometry.The IL-10 and IL-12p70 secretion levels in the supernatant were tested by ELISA method.The allogeneic T lymphocyte proliferation was tested by mixed lymphocyte reaction.The transfected cells were intravenously injected into the recipient rat on day 7 before intestinal transplantation.The survival conditions as well as pathological changes were observed in each group recipients. Results①The surface molecules CD80, CD86, and MHC-Ⅱin the PU.1 silent group were (27.0±5.6)%, (23.6±4.8)%, and (36.8±6.8)%, respectively; versus (74.0±9.4)%, (76.5±8.7)%, and (87.8±11.3)% in the negative control group, respectively, which were significantly lower in former and showing an in creased trend (P < 0.05).②Compared with the negative control group, IL-10 secretion level was significantly increased (P < 0.05), IL-12p70 secretion level significantly decreased (P < 0.05) in the PU.1 silent group.③The proliferation of T lymphocytes in the PU.1 silent group was significantly lower than that in the negative control group (P < 0.05).④When the transfected DCs were injected into the intestinal transplantation rats on day 7 before operation, the survival time was (14.3±3.3) d, (7.8±1.5) d, and (8.0±2.5) d in the PU.1 silent group, negative control group, and control group, respectively, which in the PU.1 silent group were significantly longer than that in the other two groups (P < 0.05), and the graft pathology showed that there were mild intestinal tissue damage, lymphocyte infiltration or villus edema in the PU.1 silent group. ConclusionmiRNA-155/PU.1 signaling pathway blockade could reduce DCs maturation and induce receptor-specific immune tolerance, which are proved both in vivo and in vitro.
ObjectiveTo observe the effects of Th9 cell relative factors, including PU.1, interferon regulatory factor 4 (IRF4) and interleukin 4 (IL-4), in rats with pulmonary fibrosis. MethodsNinety SD rats were randomly divided into 3 groups, ie. a normal group, a pulmonary fibrosis group, and a dexamethasone treatment group, with 30 rats in each group. Ten rats in each group were sacrificed respectively on 7th, 14th, 28th days. Model rats were induced by injecting bleomycin into trachea. Real-time PCR was applied to detect mRNA expression of PU.1 and IRF4 in bronchoalveolar lavage fluid. IL-4 in the peripheral blood was measured by ELISA. ResultsIn the normal group, the lung tissue was normal without inflammatory reaction and fibrosis at any time points. In the pulmonary fibrosis group, at the early stage the lung tissue showed alveolar inflammation with a large number of macrophages and other inflammatory cells infiltratation in the pulmonary interstitial and alveolar cavity; on 14th day, part of the alveolar structure disappeared, inflammatory cells infiltrated slightly, while the alveolar septum was mildly widened and fibroblasts proliferated; on 28th day, alveolar structure was destructed, partial alveolar walls were collapsed, alveolar septuml was significantly widened, extracellular matrix was hyperplastic, a wide range of fibrosis occured. In the dexamethasone treatment group, the alveolar structure exsisted completely, and the inflammatory cell infiltration, widened alveolar septum and fibrosis were significantly lighter than those in the pulmonary fibrosis group. PU.1 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, PU.1 mRNA were lower on 14th day and 28th day in the dexamethasone treatment group (P < 0.05). PU.1 mRNA increased from 7th day, reached peak on 14th day, and declined on 28th day. IRF4 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, IRF4 mRNA was lower on 28th day in the dexamethasone treatment group (P < 0.05). There was a positive correlation between the content of IRF4 mRNA and IL-4 on 14th day in the pulmonary fibrosis group (r=0.044, P < 0.05). ConclusionPU.1 and IRF4 play a role in inflammation leading to pulmonary interstitial fibrosis, and IL-4 may regulate Th9 cells through activating IRF4.