To investigate the role of caspase recruitment domain protein 9(card9) from macrophage in pancreatic acinar-to-ductal metaplasia.

Card9 siRNA1, card9 siRNA2 and card9 siRNA3 were constructed; fluorescence microscopy was used to investigate the fluorescence intensity of macrophages, and real-time quantitative PCR method was performed to detect the expressed level of card9 mRNA to obtain the best transfection rate. 100 μg/ml β glucan was added into 5×10⁵ macrophages in vitro culture for 12 or 24 hours, which were divided into positive group (macrophages), β glucan-stimulated positive group (β dextran+ macrophage), negative group (card9^-/- macrophage) and β glucan-stimulated negative group (β dextran+ card9^-/- macrophages). Western blotting was applied to determine the protein level of card9 in macrophages. Then, 1×10⁵ macrophages and 1×10⁵ pancreatic acinar cells were co-cultured in upper and lower transwell chamber in vitro for 120 hours, which were divided into positive group (macrophages+ acinar cells), 100 μg/ml and 500 μg/ml β glucan-stimulated positive group, negative group (card9^-/- macrophage+ acinar cell), 100 μg/ml and 500 μg/ml β glucan-stimulated negative group. Pancreatic acinar cells in the lower chamber were collected and immunofluorescence was applied to assay the duct metaplasia marker CK19 protein expression.

At 24 hours of transfection using siRNA, the intracellular fluorescence intensity in macrophages reached a peak. Card9 siRNA at the concentration of 200 nmol/l showed the highest interference efficiency. Card9 protein in positive group, β glucan-stimulated positive group, negative group, and β glucan-stimulated negative group were 0.81±0.05, 1.46±0.05, 0.42±0.06 and 0.46±0.06, respectively; card9 expression in β glucan-stimulated positive group was obviously higher than that in positive cell group, and the difference was statistically significant (P<0.05). Finally, after 100 or 500 μg/ml β glucan stimulation, the green fluorescence in pancreatic acinar cells increased significantly compared with positive group, exhibiting β glucan concentration dependence. Conversely, CK19 protein in negative group and 100 and 500 μg/ml β glucan-stimulated negative group was obviously decreased compared with positive group.

The expression level of card9 in macrophages can induce acinar-to-ductal metaplasia, indicating that card9 may mediate in the pathogenesis of pancreatic cancer.

Macrophages; caspase recruitment domain protein 9; Pancreas; Acinar cells; Ductal metaplasia