| Silicosis is an occupational lung disease characterized by the formation of silicotic nodules and pulmonary interstitial fibrosis(PIF) that results from the chronic inhalation of the dissociative silicon dioxide(SiO2) dust in the production process. Pathologically, it is characterized by continued alveolar injury, fibroblasts(FB) proliferation and a large number of extracellular matrix(ECM) deposition what caused repeated damage, repair, and leading to a large number of collagen deposition in lung tissue eventually.Previous studies showed that the incidence and development of silicotic pulmonary fibrosis is a complex and multi-stage process with the participation and interaction of a large number of cytokines, proteases and transcription factors, which is likely to involve a number of genes expression. Gene expression in the process is very complex, and the method to analyze a singly specific gene expression can not explain the complex mechanisms. Therefore, gene microarray can be used to research the coordinated groups of genes in this process as a whole, to identify the important differentially expressed genes which play a key regulatory role in the development of silicotic pulmonary fibrosis extensively. It is a new idea to explore the pathogenesis of silicotic pulmonary fibrosis further from gene level.ObjectiveSD rats were chosen as the animal model of the silicotic pulmonary fibrosis in the study by intra-tracheal perfusion of the solution of SiO2 dust, and the gene microarray was used to compare differentially expressed genes in the lung tissue between of silicotic rats and of normal ones. The differentially expressed genes of silicosis can be identified within the large-scale genome. Then a variety of methods were used for identification of differentially expressed genes, in order to find significantly related genes about silicosis, and to provide targets for effective prevention and treatment of the kind of chronic desease.Methods1. 30 of SD rats were fed adaptively in the first week, then divided into two groups randomly, the control group(6 rats) and experimental group(24 rats). The experimental group was divided into three time points such as 30d, 60d and 90d, with 8 of rats at each one.2. The experimental group was established by intra-tracheal perfusion of the solution of SiO2 dust, and the control group only received a perfusion of same amount of normal sodium. The morphologic changes of formalin-fixed, paraffin-embedded lung tissue sections tissue of rats were observed by Hematoxylin-Eosin(HE) staining. Collagens of lung tissue were stained by Van Gieson(VG)staining.3. Total RNA was extracted, then its purity was detected by ultraviolet spectrophotometer, and its integrity was tested by taking the total RNA samples to RNA formaldehyde denaturing gel electrophoresis.4. After the RNA quality check, four gene microarrays of 27K Rat Genome Array were used to identify differentially expressed genes at the time point of 60d as a whole of about 26,962 Oligo DNA of 70 mer length provided by CapitalBio Limited.5. The microarray results were compared between experimental group and control group, and differentially expressed genes were identified. Two differentially expressed genes were chosen to verify by a variety of experimental methods such as reverse transcription polymerase chain reaction(RT-PCR), immunohistochemistry(IHC) and Western Blot in lung tissue.6. The results of RT-PCR, IHC and Western Blot were analyzed with the Gel Image Analysis System, the Automatic Image Analysis System Motic Med 6.0 and Image J analysis software respectively. All the results were analyzed statistically with the software"SPSS Statistics V17.0".Results1. In the HE staining of formalin-fixed, paraffin-embedded tissue sections, the lung tissue of rats in control group at each time point displayed a slight inflammation reaction only, and there was moderate inflammation infiltration and granulomas of different sizes composed of lymphocytes, epithelioid macrophages and so on in the lung tissue of rats in the experimental group. By VG staining, the granulomas, that were silicon nodules, could be seen obviously due to the red collagen fibers.2. The ratio of OD260 to OD280 of total RNA of lung tissue in the experimental group and control group at each time point was greater than 1.9 via the ultraviolet spectrophotometer. Formaldehyde denaturing gel electrophoresis showed that all samples have clear 28S and 18S bands and a weak 5S strip. And the band of 28S is 1.5 times the intensity of 18S by visual observation.3. After microarray hybridization, total 338 differentially expressed genes were identified from the 26,962 genes (including 73 new full-length genes of rat that haven't registered in GenBank), including 267 up-regulated genes (including 67 new full-length genes of rat that haven't registered in GenBank) and 71 down-regulated genes (including 6 new full-length genes of rat that haven't registered in GenBank).4. Two significantly up-regulated genes, MMP-12 and Cathepsin E, were chosen to verify. The results of RT-PCR showed that in the lung tissue of model group at 30d, 60d, 90d, the mRNA expression of MMP-12 in the absorbance values was 4.306, 5.338, 6.713 times higher than that in the control group, meanwhile which of Cathepsin E was 1.434, 2.974, 3.889 times respectively. The results of immunohistochemical showed that in the lung tissue of model group at 30d, 60d, 90d, the protein expression of MMP-12 in the average optical density values was 1.435, 1.746, 2.069 times higher than that in the control group, meanwhile which of Cathepsin E was 1.372, 1.663, 2.103 times respectively. The results of Western Blot showed that in the lung tissue of model group at 30d, 60d, 90d, the protein expression of MMP-12 in the average optical density values was 1.214, 1.531, 1.959 times higher than that in the control group, meanwhile which of Cathepsin E was 1.262, 1.828, 1.907 times respectively. Compared with the control group, the mRNA and protein expressions of MMP-12 and Cathepsin E in lung tissue of model group were significantly up-regulated, and the up-regulating trend was the same as the result from gene microarray. All the differences were significant statistically (p <0.05).ConclusionThe experiment uses gene microarray to identify differentially expressed genes in lung tissue of silicotic rats, and further studies the molecular mechanisms of pathogenesis in silicosis. It suggested that a complex gene regulatory network may be contribute to occurrence of silicosis, and the interaction of a lot of genes may be involved in the development of silicotic pulmonary fibrosis. |
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