N of 32 g/ml was used for all further experiments. The > 자유게시판

N of 32 g/ml was used for all further experiments. The concentration of 32 g/ml is in accordance with environmentally relevant particle concentration. Higher concentrations do not reflect realistic physiological conditions [23]. We confirmed the reduced CYP1B1 mRNA-expression in additional cell types. In MDM (Fig. 4A) we observed a 2.7fold decrease of CYP1B1 after particle treatment. In sputum macrophages of healthy non smokers (Fig. 4B) we showed a 4-fold and of COPD patients a 3-fold reduction of CYP1B1. One of the COPD patients currently smoked and showed a high level of CYP1B1 mRNA in untreated sputum macrophages that was not affected after ultrafine P90 treatment. Additionally in healthy smokers no effect of particle treatment on CYP1B1 transcript level was detected. This may be due to the high concentration of organic compounds, e.g. polycyclic aromatic hydrocarbons (PAH), in cigarette smoke which in turn induced CYP1B1 mRNA expression in a competitive manner. A competitional behavior between induction of CYP1B1 mRNA by benzo[a]pyrene (BaP), a carcinogenic constituent of tobacco smoke [24], and decrease of CYP1B1 transcript by ultrafine P90 was clearly shown herein (Fig. 5). Also these findings may suggest that monocytes become less sensitive to ultrafine P90 treatment during maturation, maybe because of a stronger signal transduction or better uptake of particles in monocytes. In parallel to the lower response to particles, sputum macrophages showed no response to LPS with respect to down-regulation of CYP1B1.Epithelial cells are also affected by particle exposure. In epithelial cell lines (Fig. 4C) we observed significant down-regulation of CYP1B1 after ultrafine P90 stimulation. Primary epithelial cells obtained by bronchial brush (Fig. 4D) showed in average a 4-fold reduction of CYP1B1 mRNA with strong reductions (35- and 14-fold) in two samples. To exclude a leukocyte contamination we analyzed the cells by flow cytometry and microscopic cell differentiation. The stronger effects seen in two of the seven samples may be due to the inter-individual variation or may be due to the different clinical conditions and medications. Taken together there was no apparent clinical feature (diagnosis, medication, smoking status) to explain the higher responses of bronchial epithelial cells in the two cases. To confirm the CYP1B1 mRNA down-regulation on protein level, we isolated the microsomal fraction, because CYP1B1 is bound to the endoplasmatic membrane. It was not possible to isolate sufficient numbers of primary cells in order to obtain enough protein for Western blotting. Therefore for Western blot we used the bronchial epithelial cell line Calu-3 as a model Capivasertib cell line because they also show a decrease in CYP1B1 transcripts at mRNA level after ultrafine P90 exposure. The strongest reduction of CYP1B1 mRNA was seen after 22 h, therefore we incubated Calu-3 cell with P90 for 32 h to investigate protein expression. Densitometric analysis of the Western blots confirmed a reduction of CYP1B1 protein following P90 treatment (Fig. 6). We assume that also in monocytes and macrophages CYP1B1 protein will be decreased substantially, similar to the mRNA reduction in these PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 cells. When alveolar macrophages, monocytes, and airway epithelial cells are exposed to particles, they are phagocytized by these cell types [25,26] and can subsequently interfere with gene expression or cell functions. For cells of the monocytic lineage, one possibility is a tr.