骨桥蛋白在大鼠慢性胰腺炎中的表达及意义

Expression and significance of osteopontin in chronic pancreatitis rats

目的观察骨桥蛋白(OPN)在慢性胰腺炎(CP)大鼠胰腺组织中的表达,探讨其与胰腺纤维化的关系。方法20只雄性Wistar大鼠按数字表法随机分为对照组和CP模型组,对照组尾静脉注射无水乙醇及甘油,CP组采用单次尾静脉注射二氯二丁基锡(DBTC)构建CP模型。术后6周处死大鼠取胰腺组织,常规行病理学检查,采用Sirius-Red染色评估胰腺纤维化程度,免疫组织化学法及蛋白质印迹法检测大鼠胰腺组织OPN、α平滑肌肌动蛋白(α-SMA)及Ⅰ型胶原蛋白(ColⅠ)的表达,分析OPN表达与纤维化程度的关系。结果CP组死亡3只大鼠,成活的7只大鼠胰腺组织镜下见胰腺小叶间大量纤维组织增生,腺泡细胞空泡样变性坏死,小叶间大量炎症细胞浸润,胰管扩张。Sirius-Red染色显示CP组胰腺小叶间隙内大量鲜红色条索状及网状胶原纤维沉积,而对照组仅在血管及胰管周围有少许纤维沉积,CP组染色面积为(51±11)%,显著高于对照组的(9±4)%,差异有统计学意义(P<0.01)。免疫组织化学染色显示,CP组7只大鼠中3只胰腺组织OPN强阳性表达,4只阳性表达;对照组10只大鼠中3只胰腺组织OPN弱阳性表达,7只阴性表达,CP组OPN的表达明显高于对照组,差异有统计学意义(Z=-5.170,P<0.01),且其表达与Sirius-Red染色面积呈正相关(r=0.790,P<0.01)。蛋白质印迹法结果显示,CP组OPN、α-SMA及ColⅠ表达量显著高于对照组(0.70±0.22比0.24±0.11,0.71±0.10比0.06±0.01,2.83±1.42比0.39±0.07),差异均有统计学意义(P值均<0.05)。结论尾静脉单次注射DBTC可成功构建CP大鼠模型,CP大鼠胰腺组织OPN表达明显增加,且与胰腺纤维化程度呈正相关。

Objective To detect the expression of osteopontin (OPN) in the pancreatic tissues of chronic pancreatitis (CP) rats,and analyze the relationship between the expression of OPN and pancreatic fibrosis.Methods A total of 20 male Wistar rats were randomly divided into control group and CP model group.Control group was treated by tail vein injection of absolute ethanol and glycerol,and CP model group received a single tail vein injection of dibutyltin dichloride (DBTC) to construct CP model.Six weeks after the procedure,the rats were sacrificed to obtain pancreatic tissue,and pathological examinations were routinely performed.Sirius-red staining was used to assess the degree of pancreatic fibrosis.The expression of OPN,α-SMA and Col I in the pancreas of CP rats was detected by immunohistochemistry and Western blot,and the relationship between the expression of OPN and the degree of fibrosis was analyzed.Results In CP model group,3 rats died.In the 7 rats which survived,light microscopic examination observed great proliferation of fibrous tissue in pancreatic interlobular space,vacuolization and necrosis of acinous cells,the infiltration of inflammatory cells in the pancreatic interlobular space and dilation of pancreatic duct.Results of Sirius-red staining showed that a large number of fresh red stripe and reticular collagen fibers were deposited in the interlobular space of the pancreatic tissues in the CP group,whereas there were only a few fiber deposited around the blood vessels and the pancreatic duct in the control group and the stained area in CP group (51±11)% was significantly larger than that of control group (9±4)% and the difference was statistically significant (P<0.01).The results of immunohistochemistry showed that the expression of OPN in CP group was strong positive in 3 rats and positive in 4 rats,which in control group was weak positive in 3 rats and negative in 7 rats.OPN expression in CP group was significantly higher than that in control group (Z=-5.170,P<0.01),and the expression of OPN was positively correlated with the Sirius-Red staining area (r=0.790,P<0.01).Western-blot results showed that the expression of OPN,α-SMA and Col I in CP group was significantly higher than that in control group (0.70±0.22 vs 0.24±0.11,0.71±0.10 vs 0.06±0.01,2.83±1.42 vs 0.39±0.07),and the differences were statistically significant (all P<0.05).Conclusions A single tail vein injection of DBTC can be used to successfully establish CP rat model.The expression of OPN is significantly increased in the CP group and is positively correlated with pancreatic fibrosis.