| Background and objectiveThe studies show that fibroblasts are the main effector cells that secrete collagen fibers in liver,kidney and other fibrotic diseases,and the epithelial mesenchymal transition is one of the major sources.The pathogenesis of silicosis is not entirely clear,but it is also known that fibroblasts are the major effector cells in the synthesis and secretion of collagen fibers during the process of silicosis.Other sources of myofibroblast,as well as endogenous fibroblasts,are equally important.Alveolar type II epithelial cells have certain ability of transdifferentiation and may be one of the potential sources of myofibroblast.So far,how to transdifferentiate into myofibroblast,transdifferentiation efficiency and the time-effect relationships of transdifferentiationmarkers induced by SiO2 in the process of silicosis are still uncertain.Therefore,this study constructs the co-culture model and the experimental rat silicosis model,using transmission electron microscopy,fluorescence quantitative real-time PCR and confocal imaging and Western blot experimental technology,to explore the process and the time-effectof type II alveolar epithelial cells,which may provide a new idea of understanding the mechanism of silicosis,and provides a reference for monitoring and intervention of silicosis.Methods1.AM andAEC II were isolated from Specific pathogen-free(SPF)Sprague-Dawley(SD)rats,and the RLE-6TN was purchered from ATCC.AEC II and RLE-6TN were identified by electron microscopy.CCK-8 assay was used to determine the proper SiO2 dose by treating the cells(AM,AEC II and RLE-6TN)and with SiO2 at the concentration of 0~140μg/ mL for 24,48 and 72 hours.2.Co-culture models of AEC II were divided into the control experimental group;Co-culture models of RLE-6TN were divided into four groups(the direct control group,the direct exposure group,the indirect control group and the indirect exposure group)in vitro.To detect the expression of surface markers,including GAPDH,E-cadand α-SMAin co-culture cells by the Western blot and qRT-PCR technology.Using the enzyme linked immunosorbent assay(ELISA)determinated the TGF-βin the cell culture supernatant of AEC II and RLE-6TN.3.84 specific pathogen-free(SPF)healthy male SD rats,weight 160~180g,were purchased from the experimental animal center of Henan Province.These rats were randomly divided into control and experimental groups,6rats in each group respectively in 1week,2weeks,3weeks,4weeks,6weeks,9weeks and 12 weeks.The experimentalgroup was administrated 0.5mL silica suspension by intrareacheal instillation,Simultaneously,the control group was given saline quantitatively.HE staining was used to determine weather the model was success or not.The location and expression of E-cad andα-SMA in lung tissue were detected by immunofluorescence double staining technique.ELISA determinated the TGF-β in the lung tissue grinding fluid of rats.4.Statistical analysis: SPSS21.0 software was used to analyze the experimental data.The data were presented as the mean±standard deviation(χ ±s)if they obeyednormaldistribution.The repeated measurements and the analysis of variance w ere used in two experimental factors.Single factor data using single factor analysis of variance(one-way,ANOVA)was compared with the most significantsmall difference method(LSD)test.P value lower than 0.05 was considerd significant.Results1.Electron microscopy showed that AEC II and RLE-6TN could be seen specific lamellar body in the cell structure.The final concentration of 100 mg/mL SiO2 was chosen as the treatment dose in later experiments according to the CCK-8 assay,because of the inhibition rate reached 50% at 72 hours after exposure.2.Compared with the control group,E-cadand α-SMA relative expression levels had significant differences(P<0.05)in the primary AEC II of the experimental group.What’s more,the relative expression of E-cad andα-SMA levels showed an obvious time-effect relationship.The TGF-β in the supernatant of the experimental group had an obvious time-effect relationship and showed statistically significant(P<0.05)compared with the control group.In addition,the maximum value of TGF-βwas 1.42 ng/mL.3.Comparing with the direct control group,E-cad andα-SMA all had significant differences(P<0.05)in protein and mRNA of RLE-6TN cells in the experimental group,the same as the indirect exposed group compared with the indirect control group.In addition,We could observed the decreaseing of E-cad and the increasing ofα-SMA in protein and mRNA,and an obvious time-effect relationship in the indirect exposed group and the direct exposed group,but it was more earlier than the direct exposed groupd.The TGF-β in the cell culture supernatant of the indirect exposed group and the direct exposed group,had an obvious time-effect relationship and showed statistically significant(P<0.05)compared with their relative control groups.In addition,the maximum value of TGF-βwas 1.81 ng/mL.4.HE staining showed that a large number of inflammatory cells were accumulated in alveolar cavity and lung interval of the experimental group in 2 weeks.In 3 weeks.Especially after 3 weeks,the collagen fibers were clustered into groups,forming cell nodules and fibrous nodules in 6 weeks.After that,the nodules gradually increased,and even the pulmonary septal rupture occurred.TGF-βin grinding fluid of the experimental group reached the maximum value in 2weeks.Double immunofluorescent staining showed that the E-cad reached the maximum in 2weeks both in the control group and the experimental group,then decreased gradually,while theα-SMA showed increased gradually over time.conclusionsThe Alveolar type II epithelial cell could be transdifferentiated into myofibroblast induced by SiO2 through the epithelial mesenchymal transdifferentiation,and the alveolar type II epithelial cellwas more inclined to epithelial mesenchymal transdifferentiationin the presence of macrophages,which may be related to TGF-βsecreted by macrophages. |
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