摘要目的:探讨巨噬细胞胱天蛋白酶募集域蛋白9(card9)在胰腺腺泡细胞导管组织转化中的作用。方法:构建3条靶向card9的小分子干扰RNA(siRNA-card9)，荧光显微镜下观察转染巨噬细胞的荧光强度，采用实时定量PCR法检测巨噬细胞card9 mRNA表达量，筛选巨噬细胞的最佳转染效率。将5×10 5巨噬细胞和100 μg/ml β葡聚糖体外常规培养12、24 h后，分成阳性细胞组(巨噬细胞)、β葡聚糖刺激阳性细胞组、阴性细胞组(card9 －/－巨噬细胞)、β葡聚糖刺激阴性细胞组，采用蛋白质免疫印迹法检测各组巨噬细胞card9蛋白表达水平。取1×10 5巨噬细胞、1×10 5胰腺腺泡细胞加入Transwell小室上下层共培养120 h后，分为阳性细胞组(巨噬细胞+腺泡细胞)、100和500 μg/ml β葡聚糖刺激阳性细胞组、阴性细胞组(card9 －/－巨噬细胞+腺泡细胞)、100和500 μg/ml β葡聚糖刺激阴性细胞组，取下室的胰腺腺泡细胞，采用免疫荧光化学染色法检测腺泡细胞导管组织转化标志物CK19蛋白表达。 结果:siRNA-card9转染巨噬细胞24 h荧光强度最强，浓度为200 nmol/L时抑制效率最高。阳性细胞组、β葡聚糖刺激阳性细胞组、阴性细胞组、β葡聚糖刺激阴性细胞组培养24 h后，巨噬细胞card9蛋白表达量分别为0.81±0.05、1.46±0.05、0.42±0.06、0.46±0.06，β葡聚糖刺激阳性细胞组较阳性细胞组显著升高，差异具有统计学意义( P<0.05)。100和500 μg/ml β葡聚糖刺激阳性细胞组腺泡细胞内CK19绿色荧光较阳性细胞组明显增强，且呈β葡聚糖剂量依赖性；而阴性细胞组、100和500 μg/ml β葡聚糖刺激阴性细胞组腺泡细胞内CK19绿色荧光强度均较阳性细胞组显著降低。 结论:巨噬细胞card9表达水平升高可诱导腺泡细胞导管组织转化，提示可能存在card9介导的胰腺癌发病机制。

abstractsObjective:To investigate the role of caspase recruitment domain protein 9(card9) from macrophage in pancreatic acinar-to-ductal metaplasia.Methods: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 5×10 5 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, 1×10 5 macrophages and 1×10 5 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. Results: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. Conclusions:The expression level of card9 in macrophages can induce acinar-to-ductal metaplasia, indicating that card9 may mediate in the pathogenesis of pancreatic cancer.