八肽胆囊收缩素致急性胰腺炎体外模型的建立及其研究

目的 用CCK-8建立体外急性胰腺炎模型,并在模型中观察胰腺腺泡细胞淀粉酶、胰蛋白酶活性及F-actin的分布变化. 方法用胶原酶分离的胰腺腺泡细胞体外培养,设不同浓度的CCK-8组(10~(-12)～10~(-8) mol/L)和对照组, CCK-8刺激30 min后,用淀粉酶、胰蛋白酶试剂盒检测腺泡细胞淀粉酶、胰蛋白酶活性,用激光共聚焦显微镜观察细胞内F-actin分布的变化.结果 CK-8诱导的淀粉酶分泌率呈双峰曲线,低浓度CCK-8组(10~(-12)～10~(-10) mol/L),淀粉酶分泌率呈剂量依赖,高浓度CCK-8组(10~(-9) mol/L、10~(-8) mol/L),淀粉酶分泌被抑制;10~(-10) mol/L CCK-8时,细胞内胰蛋白酶活性明显升高(P<0.05),10~(-9) mol/L后升高更为急剧;CCK-8刺激浓度达10~(-9) mol/L后,细胞内F-actin出现重新分布,近细胞顶部区域/细胞基底部侧面的比值与对照组比较,出现显著改变(P<0.05). 结论成功建立了体外AP模型,CCK-8刺激可使胰腺腺泡细胞内F-actin分布从顶部向胞浆部、基底部变化,抑制了细胞消化酶分泌,并与腺泡细胞内胰蛋白酶的过早激活相关.

Secretagogues acute pancreatitis in pancreatic acinar cell induced by cholecystokinin

Objective The cholecystokinin-octapeptide(CCK-8)was used in the establishment of the model of acute pancreatitis in pancreatic acinar cells, and the changes of amylase, trypsin activity and the redistribution of cytoskeleton in acinar cells was observed. Methods Isolated pancreatic acini were prepared from rat pancreases by digestion with collagenase. Acini were structurally similar to those of the intact pancreas and incubated in the medium with deferent concentrations of CCK-8(10~(-12)～10~(-8) mol/L) for 30 minutes with saline as a control. Amylase and trypsin activity were measured using assay kits, F-actin distribution in pancreatic acinar cells were detected by laser confocal microscopy. Results CCK-8 generated a biphasic secretion dose-response curve in which the amylase secretion increased with the low concentrations of CCK-8 (10~(-12)～10~(-10) mol/L), and inhibited with the high concentrations (10~(-9) mol/L and 10~(-8) mol/L). The intracellular active trypsin levels was significantly increased with the dose of 10~(-10) mol/L CCK-8 (P<0.05), and elevated remarkably at the concentration of 10-9 mol/L. The redistribution of F-actin was found and the ratio of subapical/basolateral F-actin was decreased dramaticly with the high concentration of CCK (10~(-9) mol/L) in acinar cells (P<0.05). Conclusion The model of secretagagues acute pancreatitis from rat in vitro was successfully established. CCK-induced redistribution of F-actin resulted in inhibition of digestive enzyme secretion, and it is associated with the premature of trypsin in pancreatic acini.