| Objective: Severe acute pancreatitis (severe acute pancereatitis, SAP), as a kind of surgical critically ills, has high mortality, and its mechanism of occurrence and development has become the focus of surgical field. Now it has been thought that SAP refers to not only a local lesion of the pancreas, but also the systemic disease with changes in multiple organs, involving inflammatory mediators and cytokines, apoptosis, disturbance of pancreatic microcirculation and intestinal bacterial translocation, etc. Widespread concern has been attracted on the role of cytokine network and immune dysfunction in the development of SAP, mainly on the pathologic lesions due to excess inflammatory immune response and corresponding treatment. In recent years, domestic and foreign reports about the role of spleen in systemic inflammatory response involving multiple organs have increased, therefore, the research on the role of the spleen in the pathogenesis of severe acute pancreatitis is more important. This paper was aimed at observing the spleen lymphocyte apoptosis combined with other indicators in peripheral blood regarding blood amylase (AMY), endotoxin (ET), tumor necrosis factor (TNF-α), interleukin (IL-6, IL-10), and to explore the relationship between the spleen lymphocyte apoptosis and the pathogenesis of severe pancreatitis.Methods:In controlled experimental studies, 40 male or female SD rats of clean grade, weighing 300-350g (Animal Center of Hebei United University) were randomly divided into control group (sham operation group, SO group) with 10 rats and model group (SAP group) with 30 rats. Rats in model group (SAP group) were divided into three sub-groups with 10 rats in each group, and given observation every 6h, 12h and 24h respectively. Abdomen of the rat was closed after laparotomy with pancreas flipped only in sham operation group. Model rat in SAP group was given the administration of injection of 5% sodium taurocholate recessively through pancreaticobiliary duct. And then, the hole made through intestinal wall was closed with high quality sutures, and close the abdoment. The rats were killed after a certain period of time. Automatic biochemical analyzer was applied to measure amylase, and the levels regarding ET, TNF-α, IL-6, IL-10 in serum were detected through enzyme-linked immunosorbent assay. 100mg of spleen tissue from rats killed was required for detection by spectrophotometry to evaluate the activity of Caspase-3 and Caspase-9. Through TUNEL, spleen lymphocyte apoptosis can be detected and to calculate the apoptotic index (AI) and observe the ultrastructural changes of spleen through transmission electron microscope. Semi-quantitative pathological scores of pancreas can be obtained through specimens from the pancreas in rats after HE stained.Statistical analysis should be made through SPSS13.0. The data regarding ET, TNF-α, IL-6, IL-10 levels in serum and activity of Caspase-3 and Caspase-9, apoptotic index were processed through single factor analysis of variance and linear correlation analysis. Take a = 0.05 as the standard of significance test. Apoptotic index is calculated: Under high magnification(40×10), select five fields and count the apoptotic cells out of 100 cells to calculate AI.Results:1.Changes regarding blood amylase and various factors: the levels of serum AMY, ET, TNF-α, IL-6, IL-10 in the three groups of 6h, 12h and 24h were higher compared with that in the sham group with statistically significant differences (P <0.05), and it was observed that the factor in the subgroups were increased as time gose on with statistically significant differences between the three subgroups (P <0.05).2. Activity of Caspase3 and caspase-9 in spleen: activity of caspase-3 and 9 activity in the three subgroups was increased compared with that in sham operation group with statistically significant differences (P <0.05), And the activity in SAP groups was gradually increased with significant difference between the groups (P <0.05).3.Spleen apoptotic index: apoptotic cells can be seen occasionally in the sham operation group, and the amount of apoptotic cells was increased as time goes on in SAP groups with higher apoptotic index than that in sham operation group, which has significant difference (P <0.05).4.Spleen TEM: There is no obvious apoptotic cells in SO group, and apoptotic cells can be found in the subgroup of 6h with no typical apoptotic cells (apoptotic bodies), and many apoptotic cells with occasional typical apoptotic cells in the subgroup of 12h, and a great amount of apoptotic cells with typical apoptotic cells in the subgroup of 24h.5.Visual observation on pancreas: after the injection of 5% sodium taurocholate recessively through pancreaticobiliary duct, the pancreas was changed with congestion and swell within 5min, and, specimens from rats killed at certain time in subgroups of 6h, 12h and 24h peritoneal bloody ascites of different amounts , swelled pancreas with purple or violet blue color, even with yellow focal necrosis can be found, edema appears in the tissues around pancreas of different degrees. A large number of saponification plaques were formed on omentum and mesentery, gastric dilatation was also observed becoming severely with the development of disease.6.Histopathological observation on pancreas: there is no edema and necrosis of pancreatic acinus and mesenchyme in the SO group with normal lobular structure and without inflammatory cell infiltration; there appears congestion and inflammatory cell infiltration of pancreatic acinus and mesenchyme in the subgroup of 6h with lobular separation. As the development of disease, the condition of congestion and inflammatory cell infiltration of pancreatic acinus and mesenchyme becomes severe in the subgroups of 12h and with lobular separation or loss. Conclusions:1. Serum TNF-α, IL-6, IL-10 and endotoxin (ET) levels are increased significantly in the development of severe acute pancreatitis which have relationship with the occurrence and development of severe acute pancreatitis.2. Serum TNF-α, IL-6, IL-10 and endotoxin (ET) levels can be seen as an objective index to evaluate the severity and prognosis of SAP.3. Spleen lymphocyte apoptosis is related to the levels regarding TNF-α, IL-6, IL-10 and endotoxin (ET) in the development of severe acute pancreatitis and its specific mechanism needs further study.4. The activity of caspase-3 and caspase-9, as the key protease, was positively correlated with spleen lymphocyte apoptosis in the development of severe acute pancreatitis.5. There is a positive correlation between spleen lymphocyte apoptosis and the severity of severe acute pancreatitis. |
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