| Purpose:To explore the role of B10 cells in radiation-induced pulmonary fibrosis(RIPF)and whether it promotes the development of RIPF through epithelial-mesenchymal transition(EMT)and myofibroblast transformation.Method:1.Firstly establish a mouse model of radiation-induced pulmonary fibrosis,and randomly divide C57BL/6 mice into 2 groups:(1)unirradiated group;(2)simple-irradiated group.Use 18 Gy 6MV-X ray to irradiate the whole lungs to establish radiation-induced pulmonary fibrosis model.Hematoxylin-eosin(HE)staining and Masson staining were applied to observe the pathological changes,and immunofluorescence was applied to detect the expression of α-SMA protein in lung tissue to evaluate the success of the construction of RIPF model.2.Flow cytometry was taken into detection of the number of B10 cells in the lung and spleen of RIPF model at different times,so as to study the infiltration of B10 cells in the process of radiation-induced pulmonary fibrosis.3.In vitro experiments,the flow sorting technique was used to obtain B10 cells.B10 cells were co-cultured with MLE-12 cells(Mouse Type Ⅱ Alveolar Epithelial cells),taken Western-Blot,immunofluorescence and real-time fluorescence quantitative PCR(q PCR)technology into detection,which concluded the expression of epithelial markers(E-cadherin)and interstitial markers(N-cadherin)and the m RNA expression levels of interstitial cell markers(MMP9,Fibronectin 1,Vimentin etc.),to investigate whether B10 cells promote EMT in type Ⅱ alveolar epithelial cells.4.In vitro,B10 cells were co-cultured with NIH 3T3 cells(Mouse Embryonic Fibroblasts).Western blot,immunofluorescence were used to detect the protein expression of α-SMA,and q PCR were applied to detect the m RNA expression of transcription factor ZEB1 and Snail1,to explore whether B10 cells promote the transformation of fibroblasts to myofibroblasts.5.Using ionizing radiation to establish an animal model of B10 cells inhibiting radiation-induced pulmonary fibrosis,C57BL/6 mice were randomly divided into 2groups:(1)simple irradiation group;(2)irradiation + Anti-CD22 group.HE staining and Masson staining were applied to the observation in the pathological changes and fibrosis of lung tissue.Flow cytometry and immunofluorescence were applied to the detection in the number of B10 cells of RIPF model.Fluorescence method was applied to the detection of the expression of epithelial/mesenchymal cell markers and myofibroblast markers.The above methods was used to verify that B10 cells can promote the process of RIPF through EMT and induction of the transformation of fibroblasts to myofibroblasts.Result:1.After a single 18 Gy chest irradiation,a model of RIPF was successfully established.HE staining and Masson staining showed that the early(IR2d,IR14d)mice had marked inflammatory exudation in the lungs,and the late(IR 3m,IR 5m)alveolar structures were destroyed,and the interstitial collagen fibers were filled;The HE pathological scores showed statistical significance among all of time point(F =18.562,P = 0.000),and at IR 5m,the collagen deposition of Masson-stained lung was significant(t (?) = 6.006,P = 0.000).Immunofluorescence results showed that the expression of α-SMA was significantly up-regulated in the irradiation group(t =8.022,P = 0.0013).2.It was observed in the RIPF mouse model that B10 cells infiltrated in the early spleen and lung tissue.Flow cytometry results showed,compared with the non-irradiated group,B10 cells accounted for the entire spleen / lung in the early(IR2d,IR 14d)spleen(F = 22.23,P = 0.0001)and lung tissue(F = 7.485,P = 0.0044) The cell ratio increased significantly,all of which had statistical significance,while B10 cell infiltration decreased at 3 months after irradiation;and the immunofluorescence results of lung tissue B10 cells showed that the ratio of IR 2d and IR 14 d B10 cells were significantly increased compared to IR 0d(F = 9.099,P =0.0152).3.In vitro experiments,it was verified that B10 cells can promote EMT in MLE-12 cells.In the experiment of co-culture B10 cell with MLE-12,Immunofluorescence and Western-Blot results confirmed that compared with the control group,the epithelial marker(E-cadherin)was down-regulated(t=6.972,P=0.0022)and the interstitial marker(N-cadherin)up-regulated(t=4.548,P=0.01043);q PCR results showed,compared with the control group,the m RNA expression of Fibronectin1(t=7.575,P=0.0016),MMP9(t=10.18,P=0.0020)and Vimentin(t= 10.36,P=0.0005),etc.significantly increased in the B10 cell co-culture group.Similarly,the results of co-culture of B10 cell supernatant and MLE-12 cells showed,compared with the control group,the expression of E-cadherin protein was significantly down-regulated(t = 13.917,P = 0.000),while the expression of N-cadherin protein was up-regulated(t = 6.246,P = 0.003),and m RNA expression of Fibronectin(t = 6.715,P = 0.0026),and Vimentin(t = 7.88,P = 0.0014)were up-regulated.4.In vitro experiments,it was confirmed B10 cells can promote the transformation of fibroblasts to myofibroblasts.Immunofluorescence and Western-Blot results found,compared with control group,the expression of α-SMA significantly increased in the co-culture group of B10 cells and NIH 3T3 cells(t = 8.287,P = 0.0012).And q PCR results found,the m RNA expression levels of Snail 1(t = 10.26,P = 0.0005),ZEB1(t= 7.383,P = 0.0018)in B10 cell co-culture group were significantly increased;Similarly,immunofluorescence and q PCR results found that compared with the control group,the B10 cell supernatant and NIH 3T3 cell co-culture group showed the higher expression of α-SMA and significant increase of the m RNA expression levels of Snail(t = 30.35,P = 0.0001)and ZEB1(t = 3.429,P = 0.0266).5.We further used Anti-CD22 antibody to reduce B10 cells in mice,and found that it can reduce radiation-induced pulmonary fibrosis.HE and Masson staining resultsfound,compared with the simple irradiation group,the Anti-CD22 group had reduced inflammatory exudation at various time points after irradiation,and the pathological score was significantly lowered(F = 11.25,P = 0.0019);Pulmonary interstitial tissue collagen fibers hyperplasia reduced(P = 0.0002).B10 cell spleen and lung flow cytometry and lung immunofluorescence results showed,the ratio of B10 cells in the Anti-CD22 group were significantly lower at various time points after irradiation than those in the simple irradiation group,with statistical differences.Immunofluorescence results showed,in the Anti-CD22 group,the expression of α-SMA was obviously up-regulated(P = 0.0069)and E-cadherin was down-regulated(P = 0.0088)at 5months after irradiation;besides,the expression level of Vimentin was also increased at 3 months after irradiation(P = 0.0266).Conclusion:B10 cells can participate in the process of promoting RIPF by promoting the EMT of type II alveolar epithelial cells and inducing the conversion of fibroblasts to myofibroblasts. |
PDF Full Text Request