Screening Of Differentially Expressed Proteins In Rat Silicosis And Experimental Study On The Role Of S100A8 In Silicosis

Objectives This essay is to investigate the types of protein which have Significant differences in expression in the lung tissue of silicotic rats of different periods, and then picked one of the highest scoring protein(S100A8) which was validated in the animal model of silicosis and cell level making use of two-dimensional gel electrophoresis and mass spectrometry technology to prepare the rat model of silicosis in different periods and. To understand the relationship between S100A8 and silicosis, it could establish the experiment basis for researching the silicosis pathogenesis deeply.Methods 1 The replication of silicotic rats: 90 SPF male Wistar rats were fed adaptively during one week, then randomly divided into 2 groups, which had 60 rats in model group(Infection of silicosis) and 30 rats in the control group. They were instilled with Si O2 suspension through trachea without exposed, and the control group was instilled with sterile normal saline. Modeling time is 0 week, executed the rats after instilled at the 1w, 2w, 3w, 4w, 6w and 8w six time points. At the same time, observed the pathological changes in lung of rats by the naked eye and removed the lung tissue quickly. Most of them preserved in liquid nitrogen, and the remaining lung tissue was fixed to the success of modeling and immunohistochemistry to identify; 2 Screening of differential proteins of silicosis: Screened the differential protein spots by two-dimensional gel electrophoresis, and made mass spectrometry analysis to some of them; proved the differential protein S100A8 which had the highest score; 3 Validation of differential proteins of silicosis: ① Made the paraffin section of the model group and blank control group rats, and detected the S100 A by the immunohistochemistry; ② Extraction of total protein and RNA in lung tissue of the model group and blank control group rats; ③ Established the other silicosis models with rats to get alveolar macrophages, and used Si O2 to excite alveolar macrophages. After 18 hours, collected supernatant and saved for backup; ④ Model of pulmonary fibrosis in vitro replication: Got lung fibroblasts with enzyme digestion and cultured cells to the fourth generation. Fibroblasts were stimulated by the collected macrophage supernatant which has stimulated by Si O2; ⑤ Made the cell climbing piece, and detected the S100 A by the immunohistochemistry; ⑥ Extraction of total protein and RNA in fibrosis model group and blank control cells; ⑦Detected the expression of the S100A8 in silicosis model and cell levels in rats at different time points by the Western-blot and real time fluorescence quantitative PCR, and mad use of automatic image analysis system to the result of image analysis; 4 By the SPSS 16 statistical software, made the single factor analysis of variance of the experimental data, and analyzed statistically with comparison of multiple groups.Results 1 The silicosis models were copied successfully; 2 Two-dimensional electrophoresis screened 45 differentially expressed protein spots, and got 6 differential protein by spectrometry analysis from the 7 point mass, which had selected from the differential protein; 3 Immunohistochemistry and immunocytochemistry examination: the expression of S100A8 was low in the blank control group, however, expression of S100A8 in model group was higher than that of the control group, and increased gradually with time increasing; 4 The results of Western-blot assay showed that S100A8 was weakly expressed in the blank control group, however, the expression in the silicosis model of rats and cellular level was higher than that of the blank control group and gradually increased. The difference between the model group and blank control group had shown statistically significant(P<0.05); 5 Real time fluorescence quantitative PCR detection results showed that S100A8 was weakly expressed in the blank control group, however, the expression in the silicosis model of rats and cellular level was higher than that of the blank control group and gradually increased. The difference between the model group and blank control group had shown statistically significant(P<0.05).Conclusions 1 There are obvious differences of protein expression in lung tissue of rats with silicosis; 2 Through the verification of silicosis in animal models and cell level, calcium binding protein S100A8 expression was significantly different from the development of pulmonary fibrosis in rats. It may play a role in the occurrence and the development of silicosis.