胱天蛋白酶募集域蛋白9在胰腺腺泡细胞导管组织转化中的作用  

The role of caspase recruitment domain protein 9 in pancreatic acinar-to-ductal metaplasia

作　　者：李清华[1] 杨志文[2] Li Qinghua;Yang Zhiwen(Department of Gastroenterology,Songjiang District Central Hospital,Shanghai 201600,China;Department of Pharmacy,Songjiang District Central Hospital,Shanghai 201600,China)

机构地区：[1]上海市松江区中心医院消化内科,上海201600 [2]上海市松江区中心医院药剂科,上海201600

出　　处：《中华胰腺病杂志》2022年第4期278-282,共5页Chinese Journal of Pancreatology

基　　金：上海市松江区科委科技攻关项目(19sjkjgg69)

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100μg/mlβ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500μg/mlβ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500μg/mlβ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500μg/mlβ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500μg/mlβ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。Objective 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.100μg/mlβglucan was added into 5×10^(5)macrophages in vitro culture for 12 or 24 hours,which were divided into positive group(macrophages),βglucan-stimulated positive group(βdextran+macrophage),negative group(card9^(-/-)macrophage)andβglucan-stimulated negative group(βdextran+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),100μg/ml and 500μg/mlβglucan-stimulated positive group,negative group(card9^(-/-)macrophage+acinar cell),100μg/ml and 500μg/mlβglucan-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,βglucan-stimulated positive group,negative group,andβglucan-stimulated negative group were 0.81±0.05,1.46±0.05,0.42±0.06 and 0.46±0.06,respectively;card9 expression inβglucan-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 500μg/mlβglucan stimulation,the green fluorescence in pancreatic acinar cells increased significantly compared with po

关 键 词：巨噬细胞 胱天蛋白酶募集域蛋白9 胰腺 腺泡细胞 导管组织转化 

分 类 号：R73[医药卫生—肿瘤]