RESUMO
Objective To explore the effects of carboxyamidotriazole (CAI) on cytokines production by peritoneal macrophages from adjuvant arthritis(AA) rats in vitro. Methods Freund's completed adjuvant was used to induce AA in rats.Peritoneal macrophages were prepared from asepsis and incubated with CAI(10,20,40 μmol/L).The contents of TNF-α,IL-1β and IL-6 in the culture supernatant were measured by ELISA,and mRNA expressions of TNF-α,IL-1β and IL-6 were determined by real-time quantitative PCR.NF-κB p65 DNA binding activity in the nu-clear protein was detected with TransAM kit. Results The level of TNF-α,IL-1β and IL-6 in the culture superna-tant, their intracellular mRNA expression and NF-κB p65 DNA-binding activity in peritoneal macrophages of AA rats were significantly inhibited by CAI (20 and 40 μmol/L) (P<0.05 and P<0.01).Conclusions CAI may de-crease the production of pro-inflammatory cytokines such as TNF-α,IL-1β and IL-6 through inhibiting the activa-tion of NF-κB,which is potentially associated with its anti-arthritic mechanisms.
RESUMO
Objective To establish a culture method of mouse submandibular epithelial cells and to explore the optimal isolation and culture conditions so as to provide an in vitro experimental model for cell biology study and drug evaluation of salivary gland related diseases such as Sjogren's syndrome. Methods Collagenase type Ⅳ was used to digest and isolate the submandibular cells of mice. And the survival rate of cells was determined by trypan blue stai-ning. After purified by differential attachment method, the cells were cultured in F-12/DMEM medium containing10 μg/L epidermal growth factor. Optical microscope was applied to observe the morphology of the cultured cells and the cell proliferation feature was estimated by proliferation curve. In addition, immunofluorescence staining was conducted to identify the cells. Results The cell survival rate obtained by collagenase digestion was 97.5％. The morphology characteristic showed the typical epithelioid with polygon in the arrangement of typical " pebble stone" appearance. The cells were stable in growth with active proliferation according to the proliferation curve and could be subcultured to three passages. Immunofluorescence results showed that the expression of cytokeratin 8 was positive while vimentin was negative, which was consistent with the phenotypic characteristics of salivary gland cells. Conclusions The method of primary culture and subculture of mouse submandibular epithelial cells was successfully established. The method is easy to operate, which provide a potential method basis for further research study.