| Background Asbestos is one of the occupational carcinogenic factor that results in occupational mesothelioma and pulmonary cancer. In China, asbestos resource is rich and all kind of asbestos can be seen, however asbestos fibers exceed over Chinese National Standard(2mg/m3) commonly in the private enterprises and abnormity workshops. So from this point, asbestos is still one of the potential carcinogeneic factors that threaten the occupational workers. In all of the asbestos, the toxicity of crocidolite is highest than any other one. Though the pathogenesis of the malignant induced by crocidolite is complexity and is still unclear, it maybe play an important role of ROS generation and activation of EGF receptor signal pathway and MAPK cascade in the process of diseases forming induced by crocidolite.Excessive ROS causes oxidative injuries that results in the oxidation of macromolecular unsaturated fatty acid located in the biomembrane and the overexpresssion of pro-inflammation cytokine such as IL-8. Transporter NRAMP2 for divalent metal such as iron, zinc, manganese and copper can transfer the extracellular divalent ion into intercellular, its expression in epithelial cells induced by crocidolite maybe elucidate the role of iron in the process of oxidation and influence of electrontransferring between Fe3+ and Fe2+ on ROS forming.Mitogen-activated protein kinase (MAPK) is an important cascade that conducts the signal transduction in the cytoplasma. Phospho-MAPK expression showed the up-regulated manner was in alveolar type II epithelial cells stimulated by crocidolite and its phosphorylation is dependent on the activation of EGFR. Blocking the activation of EGFR lead to the inhibition of MAPK phophorylation. These data indicated that the importance of MAPK and EGFR during the process of the diseases caused by crocidolite.Objective: Crocidolite possesses the genotoxicity as shown in related publications. Research on toxicity in the cells including mesothelial cells and alveolar type II epithelial cells by crocidolite and chrysotile had done by several scientific studies in vitro, however study on the signal pathway triggered by crocidolite in human bronchial airway epithelial BEAS-2B cells has not been seen yet. Bronchial epithelial cells is also a target site for occurring cancer by crocidolite as known well, so this study on the ROS generation and EGFR, MAPK activation in BEAS-2B cells induced by crocidolite maybe disclose the partial of the signal regulation and dependence in another, the inhibition by specific tyrosine kinase inhibitors can be used to illustrate the pathogenesis of the crocidolite-related diseases, the regulation of signal pathway and the pharmacoligic strategies to find the new drugs. In addition, the inhibited phosphorylation experiments can also provide data for understanding the pathogenesis of bronchial cancer induced by crocidolite. Methods1 .BEAS-2B cells culture BEAS-2B (subclone S6) cell line was derived by transforming human bronchial epithelial cells with a SV40adenovirus construct. BEAS-2B cells grown with KGM media in the incubator at the environment of 37?C temperature and 5% CO2. Trypan Blue phagocytosis was used to evaluation of the morphology of the cells after stimulating with crocidolite. LDH kit was employed to detect the releasing of IL-8 in the culture supernatant.2.Intercellular ROS determining ROS such as O-2,OH-,H2O2 and OH-was detected using DHE and DCF fluorescence probes, at the same time, SOD reagent was employed to observe the blocking effect on ROS generation.3.Detecting Cytokine in supernatant by ELISA ELISA kit was used to detect the proinflammation cytokine IL-8 in BEAS-2B culture supernatant induced by crocidolite.4Locating the phospho-EGFR(Y1068) by fluorescent immunostaining BEAS-2B cells were seeded in Coster plate, the cells were co-cultured with crocidolite or EGF, and cells were stained using FITC-labbled antibody. The localization of phospho-EGFR (Y1068) was recognized under fluorescent microscopy.5.Western Blot5.1... |
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