ABSTRACT
Exposure to free silica induces silicosis and myofibroblasts are regarded as primary effector cells. Fibrocytes can differentiate into myofibroblast. Therefore, the present study was designed to investigate whether fibrocytes participate in silicosis. The rat model of silicosis was established. Hematoxylin-eosin stainings and Masson stainings were used to evaluate the histopathology and collagen deposition. Flow cytometry and immunofluorescence were performed to detect the number of fibrocytes and their contribution to myofibroblasts. Results showed that fibrocytes participate in silicosis. Trend analysis of different sources of myofibroblasts during silicosis indicated that fibrocytes and lung type II epithelial cell-derived myofibroblasts play an important role in the early stage of silicosis, while resident lung fibroblast-derived myofibroblasts play a predominant role during the fibrosis formative period.
Subject(s)
Animals , Rats , Disease Models, Animal , Lung , Cell Biology , Myofibroblasts , Pathology , Random Allocation , Rats, Sprague-Dawley , Silicon Dioxide , Toxicity , Silicosis , PathologyABSTRACT
<p><b>OBJECTIVE</b>The aim of this study was to investigate the effects of SiO2 on fibrocytes and whether fibrocytes participate in silicosis in vivo.</p><p><b>METHODS</b>A macrophagocyte (AM)/fibrocyte coculture system was established, and AMs were treated with 100 μg/mL SiO2. Flow cytometry was used to detect the number of fibrocytes. Real-time PCR was performed to measure the expression of collagen I, collagen III, and α-SMA mRNA. The levels of collagen I, collagen III, and TGF-β1 protein were determined by ELISA. Immunohistochemical staining was performed to measure α-SMA protein expression. A rat silicosis model was induced by intratracheal instillation of SiO2. Lung histopathological evaluation was conducted using HE and Masson's trichrome staining after 1 and 9 weeks. The number of fibrocytes in peripheral blood or lung tissue of rat was detected by flow cytometry. Double-color immunofluorescence was applied to identify fibrocytes in the lung tissue.</p><p><b>RESULTS</b>Peripheral blood monocytes were found to differentiate into fibrocytes in vitro in a time-dependent manner, and exposure to crystalline silica might potentiate fibrocyte differentiation. In addition, fibrocytes were able to migrate from peripheral blood to the lung tissue, and the number of fibrocytes was increased after SiO2 exposure.</p><p><b>CONCLUSION</b>Silica exposure potentiates fibrocyte differentiation, and fibrocytes may participate in silicosis in vivo.</p>
Subject(s)
Animals , Male , Rats , Cell Differentiation , Collagen , Metabolism , Fibroblasts , Lung , Metabolism , Pathology , Silicon Dioxide , Toxicity , Silicosis , Metabolism , PathologyABSTRACT
OBJECTIVE: To establish a genebank for phage single-chain antibody for further screening the specificity of single chain fragment variable( Sc Fv) in lung tissue of silicosis rats by phage display technology. METHODS: Twenty-four specific pathogen free male SD rats were used to construct silicosis model by one-time bronchial perfusion with 1. 0 m L of silicon dioxide suspension( mass concentration,100 g / L). We took periphery blood from 6 rats 3,6,9 and 12 weeks respectively after establishing the model. The peripheral lymphocytes were mixed,and total RNA was extracted using Trizol,and c DNA was synthesized by reverse transcription. The degenerated primers were used to amplify the variable region of heavy chain( VH) gene and variable region of light chain( VL) gene by polymerase chain reaction( PCR).Then VH and VL genes were assembled to form Sc Fv by T4 DNA linker. The cloning recombinant of Sc Fv and plasmid of PCANTAB-5e were transformed into competence E. coli TG1 by calcium chloride. The Sc Fv genebank of silicosis model was constructed by M13K07 helper phage superinfection. There were 10 bacterial colonies for plasmid restriction dualenzyme digestion randomly selected for confirmation. RESULTS: Agarose gel electrophoresis showed that there were two bands of obvious 28 S and 18 S in total RNA of periphery blood lymphocytes of silicosis rats. The total RNA was intact. The size of VH gene fragment was about 400 bp,the size of VL gene fragment was about 350 bp and recombinant Sc Fv gene fragment length was about 750 bp. The helper phage was amplified and placed with double-deck agar plate and observed limpid plaque with the size of a rice grain. The phage titer was 1. 35 × 10~(16) pfu / L. The recombinant plasmids were transformed into E. coli TG1 and total bacterial count was 8. 0 × 10~9 cfu / L in resistant plate. The positive cloned plasmid PCR gel electrophoresis and double enzyme results showed a positive inserting rate of 90. 0%. The capacity of phage single-chain antibody genebank of experimental silicosis was 7. 2 × 10~9 cfu / L. CONCLUSION: The silicosis rat model with phage Sc Fv gnebank could be successfully established,and its capacity and diversity provide support for the follow-up screening.
ABSTRACT
Pure culture of Phlebopus portentosus was inoculated in the roots of coffee tree. The results indi-cated that the young fruit bodies would come out around the rhizomes of host tree after inoculation in 30 to 90 days, single or cluster, 3 to 4 days for mature, weight 20.0 g to 62.0 g. Brown rhizomorph and hyphae can be seen on the seedlings`rhizome, main root and side root while nothing is on the tip of the root.It was found that rhizomorph on the surface of roots would die after inoculation in 90 days in pot.