| According to statistics, 2/3 of all occupational diseases in China are the silicosis, which is one of the most risk occupational diseases in our country at present. On the basis of previous studies, the occurrence and develop- ment of silicosis is a multi-stage process with participation and interaction of many molecules and cells. Related genes have played different roles in the occurrence and development of silicosis. These genes, however, might cooperate with each other to determine the occurrence and development of silicosis in different time and space. Therefor, to study the silicosis by means of modern molecular biology, especially to define related genes as a gene cluster, and to select key genes of silicosis is important to deeply study molecular mechanisms and to explore health monitoring measures, early diagnosis methods, and new therapy means of silicosis.Professor Wang has been engaged in molecular pathology of fibrosis for long-term, and has found relationship of pulmonary fibrosis of silicosis and IL-lβ, TNF-α, TGF-β1, NF-κB, PDGF, etc. And these results suggested gene expression did have some changes in the occurrence and development of silicosis. However, most of these studies analyzed a single gene or a few genes isolatedly, and didn't fully reflect relationship between structural differences or expression differences of genome and silicosis. With the rapid development of biotechnology, humanity has entered a post-genome era, and the focus of researches will shift from discovering genes to discovering function of genes. The technique of gene microarray, with large-scale and high-throughput analysis to gene expression, can be used to the comparative study for the complete gene expression profiles in the pathogenesis of silicosis. And we can identify related genes of pulmonary fibrosis of silicosis with gene microarray.Funded by Natural Science Foundation of Hebei Province, this study uses 35K Human Genome Array of CapitalBio Corporation, and has initially identified candidate differentially expressed genes , which are related to the pathogenesis of pulmonary fibrosis of silicosis in people. Bioinformatics classification tools are combined with to classify function of genes and to analyze their genes biological pathways.Objectives1 To compare to gene expression profiles of lung fibroblast between in a cellular model of silicosis in vitro and in a normal celluar model in vitro, and to identify differentially expressed genes.2 To classify the bioinformation of these differentially expressed genes.3 To test expression of IGFBP-5 (up-regulated gene) and MMP-3 (down- regulated gene) in vitro cell model of silicosis, which verifies the reliability of results of gene microarray.Methods1 To identify related genes of pulmonary fibrosis of silicosis1.1 With the method of cell culture in vitro, AM (alveolar macrophages) of silicotic patients , cell lines of THP-1 and HFL-I were utilized as experimental materials.1.2 Human AMs were collected from a silicotic patient by bronchoalveolar lavage (BAL) and exposed in the presence of SiO2 (50μg/ml) for 18h, then these conditioned supernatants were collected.1.3 Cell lines of THP-1 were exposed in the presence of PMA (160 nmol/L)for 48h, when suceeded, blank DMEM would be changed to culture these stimulated cells, and these supernatants were collected after 18h.1.4 The experiment was divided into two groups: AM supernatants were used to incubate HFL-I to make a cellular model of silicosis in vitro (group S); PMA-stimulated THP-1 cells supernatants were used to incubate HFL-I to make a cellular model of normal lung in vitro (group C).1.5 Supernatants of two groups were incubated with HFL-I for 36h, and then we extracted the total RNA, which would be sent to CapitalBio Corporation to identify differentially expressed genes. 2 To classify the bioinformation of these differentially expressed genes:The MAS 3.0 system was used to classify the bioinformation of these differentially expressed genes.3 To verify partial results of gene microarray by using technique of Immunocytochemistry(ICC), Western-blot and RT-PCR3.1 The protein expression of IGFBP-5 and MMP-3 were authenticated in both groups with the method of ICC and Western-blot , and incubation time points were 6h, 12h, 24h, 36h, 48h, 72h.3.2 The mRNA expression of IGFBP-5 and MMP-3 were authenticated in both groups with the method of RT-PCR, and incubation time points were 6h, 12h, 24h, 36h, 48h, 72h.4 Results were analyzed semiquantatively with the Automatic Image Analysis System. And all results were analyzed statistically with the software"SPSS 13.0".Results1 Differential gene experssion profiles: According data analysis, this sutdy got 8999 effective genes, and 84 identified genes, including 43 commonly up-regulated genes and 41 commonly down-regulated genes.2 Results of Bioinformatics classificationBased on three kinds of GO ontology provided by the GO database of MAS3.0 system, we categoried differentially expressed genes, and got 107 GO functional clusters, mainly involving molecular functiones of the cell cycle, cell differentiation and proliferation, apoptosis, cytoskeleton, cell signal transduction, transmission, oxidation stress, response, transcription regulation, etc; Based on KEGG database of MAS3.0 system, we analyzed differentially expressed genes, and got 31 signal transduction pathways, mainly related to P53 signaling pathway, Jak-STAT signaling pathway, PPAR signaling pathway, TGF-βsignaling pathway, Toll-like by body signaling pathway, polymerization adhesive, calcium signaling pathway, purine metabolism, pyrimidine metabolism, the renin-angiotensin system, etc.3 Results of verifing the gene microarray with ICC, Western-blot and RT- PCR methods:3.1 Protein and mRNA expression of IGFBP-5 in group S and group C: Protein and mRNA expression of IGFBP-5 were higher in group S than Group C at each time point, and differences were statistically significant at 12h, 24h, 36h, 48h and 72h. These results were consistent with results of gene microarray.3.2 Protein and mRNA expression of MMP-3 in group S and group C: Protein and mRNA expression of MMP-3 were lower in group S than group C at each time point(except 6h ), and differences were statistically significant at 12h, 24h, 36h, 48h and 72h. These results were consistent with results of gene microarray.Conclusions:1 It is rare at home and abroad that analyzing gene expression profiles in pulmonary fibrosis of human silicosis with gene microarray. The study has selected 84 differentially expressed genes, including 43 genes were commonly up-regulated and 41 genes were commonly down-regulated. These results suggest that abnormal expression of multiple genes is existing in the occurrence and development of pulmonary fibrosis, and these genes provide lots of valuable information for the research of molecular mechanisms of pulmonary fibrosis.2 Clustering functione and analyzing signaling pathways systematically, may promote the re-consideration for the occurrence and development of silicosis on the eye of systematic biology.3 Selecting IGFBP-5 (up-regulated gene) and MMP3 (down-regulated gene) from results, and using technique of ICC, Western-blot and RT-PCR, we test both protin and mRNA expression in two groups. Results agree with gene microarray results basically, which furtherly confirm the reliability of results of gene microarray. |
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