持续抑制超氧歧化酶活性诱导大鼠慢性胰腺炎模型

Sustained suppression of superoxide dismutase activity induces chronic pancreatitis in rat

目的观察大鼠腹膜内注射超氧歧化酶（SOD）抑制剂二乙基二硫代氨基甲酸盐（DETC）后胰腺的病理改变，并与大鼠胰管内注射三硝基苯磺酸（TNBS）所制备的慢性胰腺炎（CP）模型相比较。方法将大鼠按随机表法分为DETC组、DETC对照组、TNBS组、TNBS对照组、正常对照组。DETC组大鼠腹膜内注射DETC750mg／kg体重，每周2次，DETC对照组在腹腔内注射等容积生理盐水。TNBS组大鼠胰管内注入含2％TNBS的乙醇磷酸盐缓冲液，TNBS对照组注入等容积乙醇磷酸盐缓冲液。正常对照组不做任何处理。术后2、4、6、8周分批处死大鼠，取血检测淀粉酶活性，取胰腺组织行病理及超微结构检查，检测组织内SOD、谷胱甘肽过氧化物酶（GSH-PX）活性和丙二醛（MDA）含量，免疫组化法检测组织α-平滑肌肌动蛋白（α-SMA），结蛋白（Desmin），胶原Ⅰ、Ⅲ，TGF-β1，纤维连接蛋白（FN）的表达，RT-PCR法检测组织TGF-β1mRNA表达。结果DETC组大鼠无死亡，TNBS组大鼠死亡率为15％。2组大鼠血淀粉酶活性差异无统计学意义。4周时DETC组大鼠胰腺纤维化评分为（3．4±1．1）分，显著高于TNBS组的（3．0±1．3）分（t=3．462，P〈0．05）；6周时胰腺组织腺体破坏评分为（9．1±1．8）分，显著高于TNBS组的（8．4±1．8）分（t=2．943，P〈0．05）；细胞空泡样变、脂肪浸润评分较TNBS组高，但差异均无统计学意义。DETC组和TNBS组在制模2周后即可见胰腺超微结构改变，4周后可见大量新生或已趋成熟的胶原纤维。2周时DETC组SOD活性较TNBS组显著下降（t=5．468，P〈0．05），GSH．PX活性在2、6周时较TNBS组显著下降，（t值分别为6．497，10．125，P〈0．05），而MDA活性在6、8周时较TNBS组均显著升高（t值分别为3．350，5．407，P值〈0．05）。DETC组和TNBS组大鼠胰腺组织α-SMA，Desmin，胶原Ⅰ、Ⅲ，TGF-β1和FN表达及TGF-β1mRNA表达水平差异均无统计学意义。结论应用DETC持续抑制SOD活性可成功诱导CP。DETC组大鼠的胰腺脂肪浸润和纤维化程度较TNBS组出现得更早、更严重。采用该法制模操作简单，大鼠死亡率低，是一种较理想的制备CP模型的方法。

Objective To investigate the pathologic changes in the pancreas of rats after intraperitoneal injection of DETC, a kind of superoxide dismutase （SOD） inhibitor, and to compared that with another model of chronic pancreatitis by pancreatic duct injection of TNBS. Methods The rats were randomly divided into DETC group, DETC control group, TNBS group, TNBS control group, normal control group. Rats in DETC group received an intra-peritoneal injection of DETC twice a week, and rats in DETC control group received an intra-peritoneal injection of same amount of normal saline. Rats in TNBS group was injected with 2% TNBS ethanol phosphate buffer into the pancreatic duct, while rats in TNBS control group was treated withinjection of same amount of ethanol phosphate buffer, and rats in normal control group received no treatment. The rats were sacrificed after 2 w, 4 w, 6 w and 8 w. The serum levels of amylase were determined, and pathological and uhrastructure changes of the pancreas were measured. The levels of SOD, GSH-PX activity and MDA content were detected. The expressions of α-SMA, Desmin, Collagen Ⅰ , Collagen m, TGF-β1, FN in tissue were detected by immunohistoehemical assay. The TGF-β1 mRNA expression was detected by RT- PCR. Results No rat died in DETC group. The mortality rate of TNBS group was 15%. The serum levels of amylase were not statistically different between the 2 groups. The fibrosis scores of rat in DETC group at 4 w was 3.4 ±1.1, which was significantly higher than that in TNBS group （3.0 ±1.3, t = 3. 462, P 〈 0.05 ）. At 6 w, the damage scores of rat in DETC group was 9.1± 1.8, which was significantly higher than that in TNBS group （ 8.4 ± 1.8, t = 2. 943, P 〈 0.05 ）. Scores of vacuolar degeneration and fatty infiltration of rat in DETC group were higher than those in TNBS group, but the difference between the two groups was not statistically significant. Two weeks later, uhrastructure changes of pancreas could be observed, and large amounts of regenerative or mature collagen could be seen at 4 w. The SOD activity of DETC group was significantly decreased when compared with those in TNBS group （t =5. 468, P 〈0.01 ）. The GSH-PX activity of DETC group at 2 w, 6w was significantly decreased when compared with those in TNBS group （ t =6. 497, 10. 125, P 〈0.01）. While the activity of MDA at 6 w, 8 w was significantly increased when compared with those in TNBS group （t =3.350, 5.407, P 〈0.05 ）. The differences at other time points were not statistically significant. The expressions of α-SMA, Desmin, Collagen Ⅰ , Collagen Ⅲ, TGF-β1, FN, and TGF-β1 mRNA were not statistically significant between the 2 groups. Conclusions Sustained suppression of SOD activity can successfully induce chronic pancreatitis. Fatty infiltration and fibrosis in pancreas in DETC group occurs earlier with more severe presentation than that in TNBS group. Intraperitoneal injection of DETC is easy with low mortality rate, which is an ideal method for chronic panereatitis model induction.