Expression Of Cytokines In Pneumoconiosis And Their Significance

The hazard of Pneumoconiosis is severe in our country, which is still in higher incidence rate and there has been no effective therapeutic tool till now. It's crucially and practically significant for improving prewarning, health surveillance, biological monitoring and updating prevention level to screen biomarker which can reflect workman's exposure and response level. Mass data indicate that many kinds of cytokines are relative to the development process of pneumoconiosis, during which transforming growth factor beta 1 (TGF-β1), platelet derived growth factor(PDGF) and connective tissue growth factor(CTGF) play crucial roles in regulating fission and proliferation of fibroblast and synthesis and degradation of collagen protein.Transforming growth factor betal (TGF-β1) is a kind of pleiotropy polypeptide, which biologic activity is very extensive, including promote interstitial substance cell proliferation, cell differentiation and migration etc. Such as promoting fibroblast proliferation, increasing fibroblast secretes extracellular matrix(ECM) etc. It is clear that TGF-β1 participates in the process of fibrosis, such as lung fibrosis, liver fibrosis, kidney fibrosis and dermatofibrosis, research team that researcher placed discovered that TGF-β1 had close relation with the development of coal worker' pneumoconiosis(CWP); Connective tissue growth factor (CTGF) is up-regulated in a variety of fibrotic disorders, probably secondary to the activation and production of transforming growth factor-betal (TGF-β1).In a rat wound-healing model, Inkinen K discovered that fibroblast-like cells expressed CTGF most intensively in situ hybridization and CTGF expression together with TGF-β1 and PDGF are up-regulated in wound healing, the expression of CTGF mRNA in Northern blot and immunohistochemistry were correlated to the expression of TGF-β1 and platelet-derived growth factor (PDGF); TGF-β1 modulates connective tissue growth factor expression via Smad2 signaling pathway in podocyte in vitro. These research suggest that TGF-β1, CTGF and PDGF had close relation with the development of fibrosis. Research on TGF-β1 and organ fibrosis have had more report, and research on TGF-β1 and pneumoconiosis have had some report, but its concrete mechanism is not clear. While the relation of CTGF and PDGF and organ fibrosis has been some reported, but the relation of them and pneumoconiosis has not been yet reported.In present studies, we performed reverse transcription PCR, immunohistochemistry and enzyme-linked immunosorbentassay(ELISA) to measure the mRNA and its coding protein levels of CTGF, TGF-β1 and PDGF and analyze the relationship between these three cytokines and pneumoconiosis, and then to analyze the potential role of CTGF, TGF-β1 and PDGF during mere influrence on the development of pneumoconiosis and approach cytokines on the mechanism of pneumoconiosis development, in order to offer theoretic base on exposure and effect biomarker of pneumoconiosis, prewarning and health surveillance. 1 Expression of CTGF, TGF-β1 and PDGF in rats' pneumoconiosis and their significance1.1 Experimental model establishment:40 SPF healthy adult rats supplied by experimental animal center in Henan province were adopted and their certificate number is 410117 and body weight is 185～212g. And then the suspension of silica dust and animal experimental model was prepared. After exposed to silica dust for 45 days, rats were sacrificed and taken out lung tissues that were washed then using physiological saline. The bulk of lung tissues were preserved in nitrogen canister and tithe of them were fixed by 10% neutral formalin.1.2 Methods:Total RNA was extracted from lung tissues of rats by using total RNA kit, and then the content and purity were evaluated by 1 % agarose gel electrophoresis(AGE) and the OD value of RNA . Total RNA was progressed directly to cDNA by reverse transcription using AMV reverse transcriptase produced in Shanghai Sangon bioengineering technical service company. The present study performed reverse transcription PCR and immunohistochemistry to measure and analyze the mRNA and its coding protein levels of CTGF, TGF-β1 and PDGF. Statistical analysis: Data was expessed by mean±standard deviation, group comparision adopted independent-samples t-test, the correlation among three cytokines adopted Pearson correlation; P value less than 0.05 was considered significant. Biosens Digital Imaging System was used in the half quantative analysis of the immunohistochemical results analyzed by independent samples t-test, the correlation among three cytokines adopt Pearson correlation; P value less than 0.05 was considered significant.1.3 Results:From the results of the expression of mRNA and its coding protein, the expression of CTGF, TGF-β1 and PDGF mRNA level were higher in the experimental group compared with the control group. The correlation among the expression both in mRNA and its coding protein of CTGF, TGF-β1 and PDGF was positive.2 Expression of CTGF, TGF-β1 and PDGF in pneumoconiosis patients' serum and their significance2.1 Subjects:Cases: 5 cases 0 plus without pneumoconiosis and 65 cases of pneumoconiosis were selected based on GBZ70-2002 and investigated by questionaire, including 24 cases of silicosis and 41 cases of pneumoconiosis. All subjects were males and Han race and workers without other pulmonary or autoirnmune diseases. Their average age was 65.50±5.44 years old, exposure time limit is 26.41±6.38 years, main type of work in production were mining and tunnelling etc.Controls: 77 cases of healthy males were selected, and all subjects had no dust exposure vocational history without other organ fibrosis.their average age was 60.08 + 8.24 years old.2.2 Methods:Questionaire: all subjects accepted questionaire, including individual general state of health, dust exposure history, pneumoconiosis diagnosis history, previous medical history and family medical history etc;Blood sample collecting: all subjects were collected blood sample on an empty stomach.TGF-β1, CTGF and PDGF content of serum detecting : Use the elisa reagent kit to detect TGF-β1, CTGF and PDGF content of serum .According to the direction , the operation was finished .The data was processed by means of SPSS 13.0 software . P value less than 0.05 was considered significant.2.3Results: The serum level of TGF-β1, CTGF and PDGF in 0 plus group without pneumoconiosis were partly higher than those in each stage of pneumoconiosis and in the control group; The serum level of TGF-β1 and PDGF in the patients with pneumoconiosis were higher than those in the control group; The serum level of TGF-β1 and PDGF in the patients with pneumoconiosis decreased with the increasing of pneumoconiosis stage, while the serum level of CTGF in the patients with pneumoconiosis increased with the increasing of pneumoconiosis stage. The bivariate correlation between the serum level of TGF-β1 and PDGF in the whole objects was positive.3 Conclusions:3.1The results suggests that it's possible for CTGF, TGF-β1 and PDGF participating in the development process of pneumoconiosis, and it offers significant biological parameter on prewarning and health surveillance of pneumoconiosis.3.2The serum level of TGF-β1, PDGF and CTGF in the patients with pneumoconiosis might correlate during the nosogenesis and exposure on different dust type.