糖原合酶激酶-3B在大鼠急性水肿型胰腺炎模型中的表达变化及意义

Expression of Glycogen Synthase Kinase-3B in Pancreatic Tissue of Acute Edematous Pancreatitis Rats

目的探讨糖原合酶激酶-3B(GSK-3B)表达与急性水肿型胰腺炎(AEP)严重程度的相关性。方法将36只雌性Wistar大鼠随机分为:生理盐水(NS)对照组,AEP组(又分为造模后1、3、6、12、18 h组),每组6只。AEP组腹腔内注射雨蛙素建立AEP模型,NS对照组腹腔注射相同剂量的生理盐水。ELISA法检测血清胰淀粉酶、脂肪酶水平,测定胰腺干湿比重,RT-PCR分析GSK-3B及caspase-3、9 mRNA表达变化,Western blot检测GSK-3B蛋白表达,免疫组化检测GSK-3B在胰腺组织中的表达定位,TUNEL法检测胰腺腺泡细胞凋亡。结果成功建立大鼠AEP模型,胰腺组织病变程度于造模后6 h最严重;GSK-3B在胰腺组织的腺泡及内皮细胞有表达,定位于胞浆;AEP组GSK-3B、caspase-3、9 mRNA及GSK-3B蛋白与对照组相比,表达增加(P<0.05),并于造模后6 h达高峰(mRNA于造模后3 h达高峰);AEP组胰腺腺泡细胞凋亡指数高于对照组(P<0.05),亦于造模后6h达高峰;腺泡细胞凋亡指数与血清胰淀粉酶、脂肪酶,胰腺干湿比重,GSK-3BmRNA表达呈正相关(r=0.71,0.74,0.89,0.94,P<0.05),GSK-3BmRNA与caspase-3,9 mRNA表达呈正相关(r=0.73,0.71,P<0.05)。结论 GSK-3B表达随着大鼠AEP严重程度加重及腺泡细胞凋亡增多而升高,与疾病严重程度基本一致。

Objective To investigate the expression of apoptosis related gene glycogen synthase kinase-3B（GSK-3B） in acute edematous pancreatitis（AEP）,and the relationship between GSK-3B and AEP.Methods The AEP rat model was established by the injection of caerulein（50 μg/（kg·h）） intraperitoneally per hour for 4 times.There were total 36 rats,every 6 rats were sacrificed at 0 h,1 h,3 h,6 h,12 h and 18 h after the injection of caerulein.Blood samples were taken from portal vein for ELISA examination of pancreatic amylase and lipase,pancreatic mRNA was extracted for RT-PCR analysis of GSK-3B,caspase-3,9.The expression of GSK-3B protein was examed by Western blot,and acinar cell apoptosis was measured by TUNEL assay.Results AEP was successfully induced and pancreatic lesions were most severe at 6 h after the injection of caerulein.Immunohistochemistry assay showed that GSK-3B protein located in cytoplasm of pancreatic acinar cells and endothelial cells,but not in islet cells.The mRNA levels of GSK-3B,caspase-3,9,the protein level of GSK-3B,as well as cell apoptotic index in AEP rats were significantly increased when compared with the control rats received normal saline.Acinar cell apoptosis index was positively correlated with serum amylase,lipase,ratio of dry/wet of pancreas and GSK-3B mRNA expression,while GSK-3B mRNA level was positively correlated with caspase-3,9 mRNA expression.ConclusionThe expression levels of GSK-3B mRNA and protein were related to the severity of AEP and acinar cell apoptotic index.