| Objectives:Through experiment the model of the rats liver Ischemia reperfusion, observing in the liver ischemia-reperfusion the protection of ischemic postconditioning on bile duct cells.Explore the mechanism of inhibition of bile duct apoptosis in ischemic postconditioning on liver ischemia-reperfusion injury, providing more effective measures and mechanism theory for relieving biliary injury in clinical liver ischemia-reperfusion.Methods:1. Experimental model:Will be 24 healthy male rats,Weight is 200 g to 300g, randomly divided into sham-operation (SO) group;Ischemia reperfusion (IR) group;Ischemic postconditioning (IPO) group,8 in each group.Groups of SD rats adaptive feed one week, preoperative fasting 12 hours, free water. Take 0.3% pentobarbital sodium 30 mg/kg intraperitoneal injection of anesthesia, after the success of anesthesia, to fix the rats in the operating table, cut the hair and disinfected. All procedures are under aseptic conditions.1).sham-operation (SO group):after anesthesia, cut the hair and disinfected, success with Single sterile, on the midline take a Longitudinal incision about 3-4 cm long (make liver fully shown), cut open the abdominal wall layers, show the liver, duodenohepatic ligament was dissected only, close abdomen, without additional intervention.The purpose is as normal control group to rule out the interference of operation factors on experimental results.2).Ischemia reperfusion group (IR):on the basis of SO group, after dissection of the duodenohepatic ligament,simulating Pringle method, clip the hepatic portal area with no damage arteries clip, cause liver ischemia, after 30 min take down the vascular clamp to restore blood supply, Continuous reperfusion of liver.close abdomen.3).The ischemic postconditioning group (IPO) based on IR group, before fully recover blood supply, achieved by10-second reperfusion followed by 10-second reocclusion for six times (2 min), then fully recover blood supply ?2.Collect the specimen of groups of rats four hours after operations, drawing heart blood by blood collector,standing for 30 min at the room temperature, and then put in a centrifuge 3000 r/min, the centrifugal 15 min, after the completion of the centrifugal extraction serum specimens, deposited in the-80 ? refrigerator, superoxide dismutase (SOD) levels and r-glutamyl transpeptidase (r-GT) activity to be detected. Draw liver tissue specimen in the hepatic portal at the same time,put in the 10%neutral formaldehyde and fixed for 24 hours, paraffin embedding, for detection.3.The level of SOD and r-GT was measured by colorimetric method.4. Observe and compare the changes in the bile duct cell morphology by HE staining;5.Detect the expression levels of BCL-2 and the Bax in bile duct cells by immunohistochemical method;6.Terminal-deoxynucleotidyl transferase mediated d-UTP Nick end labeling (TUNEL) to detect bile duct cells apoptosis, and calculate the apoptosis index (AI).7.The data were analyzed by SPSS 13.0, The results expressed in mean±standard deviation (x± s) said,use SNK method of analysis of variance to compare multi-group data..P<0.05 means there were statistical significance in these groups.Results:(1).HE staining showed:HE staining showed:SO group hepatocyte in the liver tissue are integrity, lobular structures are no damage, Portal area is nomal, and there is no abnormal in structures of Bile duct, no or only a small number of neutrophil infiltration. In the IR group, the part of lobular in the liver has no clear structure, a large around the central vein of liver cell necrosis.The epithelial cells of portal area are damaged,and with a large number of neutrophil infiltration,intrahepatic bile duct epithelial cells fall off. more cell apoptosis. IPO pathological changes reduce significantly compared with ischemia-reperfusion group, lobular architecture is complete, liver cell denaturation degree ease, portal area inflammatory infiltrates lighter, biliary epithelia continuous complete, inflammatory cells infiltration lighten.(2).The r-GT which reacts the bile duct epithelial cells injury has minimum activity in the control group, the IR group activity is highest, IPO activity is higher than SO group, and that decreased the IR group, and all the differences were statistically (P< 0.05).(3). Detect the expression of serum SOD levels showed no significant difference between the expression of SOD in each group,but the difference was not statistically (P>0.05).(4). The expression of antiapoptosis BCL-2 protein:SO group of bile duct tissues the BCL-2 positive expression rate were significantly lower than the IR group and IPO group, and the difference was statistically (P< 0.05). IR group of bile duct Bcl-2 positive expression rate was lower than that in group IPO, the difference was statistically(P<0.05).(5). The expression of promoted apoptosis Bax proteins:IR group and IPO group Bax positive expression rate of was higher than that of SO group, the difference was statistically (P<0.05), and the IPO of the positive expression rate is lower than the IR group,the difference also was statistically(P<0.05).(6).The level of bile duct tissue cell apoptosis:SO group of bile duct tissues only visible a small amount of apoptosis cells express, apoptosis index was 26.06±2.27, and the apoptotic cells in IR group expression enhanced apoptosis index was 79.31 ±1.12, comparition of the two AI the difference was statistically (P<0.05), IPO group apoptosis index was 58.90±2.75, and apoptotic cells in IPO group decreased in comparison with the IR group, AI decreased, and the difference was statistically (P<0.05), but not fall to the SO group level, compare the AI of the two group, the difference was statistically (P<0.05).Conclusions:1.The liver ischemia-reperfusion can lead to biliary epithelial cells apoptosis, causing bile duct injury.2.Ischemic postconditioning can relieve the liver and bile duct tissue damage in ischemia-reperfusion injury.3.In the case of not to interfere in bile duct tissue blood flow, can not cause the change of the bcl-2 protein and Bax protein.4.Ischemic postconditioning may protect biliary epithelial cells by promoting the bcl-2 protein expression and inhibiting the expression of Bax protein, and that inhibit the happening of the bile duct cell apoptosis. |
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