| Objective: In order to evaluate the role of hepatic Kupffer cell (KC) on the high incidence rate of organ injuries and high mortality rate, and the mutual relations between hepatic Kupffer cell to the degree of SIRS in acute pancreatitis course, animal experiments and primary cell cultures were used respectively to observe the effects of hepatic Kupffer cell on acute lung injury during acute pancreatitis. It is hoped that this study can devote something to improve the treatment effect of acute pancreatitis, reduce the incidence rate of severe acute pancreatitis (SAP) and decrease the death rate in clinic.Methods: 1. In experiment 1, the health male Wistar rats were randomly divided into 3 groups. The animals in group A were retrogradely injected 5% sodium taurocholate into the pancreatic ducts to establish acute necrosis pancreatitis models (ANP group). In group B, the ANP models were established after Kupffer cells were inhibited previously by intravenous gadolinium chloride (inhibited KC group). The rats in group C were given gadolinium previously to inhibit Kupffer cells, and then physiological saline was injected retrogradely into the pancreatic ducts (control group). The observation items were: levels of serum amylase, pancreatic morphological changes, concentrations of serum TNF-a and IL-1 P , pulmonary morphological changes, wet/dry weight ratio of lung tissue (W/D ratio) and changes of partial pressure of oxygen in arterial blood (PaCh). 2. To fit in with the requirement of experiments, we isolated, cultured and identifyed hepatocytes, Kupffer cells and pulmonary microvascular endothelial cells (PMVEC) for getting cells with high purity and activity. 3. In experiment 2, Kupffer cells were divided into 4 groups. In group A, physiological saline was added into the supernatant fluid of media as control (control group). Lipopolysacchride (LPS) was used in group B (LPS group). In group C, pancreatic elastase was added (elastase group). And group D was treated with both lipopolysacchride and pancreatic elastase (LPS+elastase group). The expression of TNF- a , IL-1 3 and TLR4mRNA in Kupffer cells were determined by RT-PCR, concentrations of TNF- a and IL-1 P in the supernatant fluid of media by ELISA. And Western blot was used to observe the expression changes of TLR4 protein in Kupffer cells. 4. In experiment 3, the cultured PMVECs weredivided into 3 groups. The supernatant fluid of cultured Kupffer cells pretreated with LPS and pancreatic elastase was added into group A (KC group). The supernatant fluid of cultured hepatocytes pretreated with LPS and pancreatic elastase was added into group B(hepatocyte control group). The supernatant fluid of cultured Kupffer cells without LPS and pancreatic elastase pretreated was added into group C (control group). The morphological changes of PMVECs under microscope were observed, the changes of F-actin and the concentrations of [Ca2+]i in PMVECs were determined.Results: 1. In experiment 1, no significant differences were observed in serum amylase between the animals of group A (ANP group) and B (inhibited KC group), however, the levels of serum amylase in group A and B were significantly higher than that in group C (control group). The morphological changes of pancreas in group A and B fitted in with the changes of ANP, while no relative changes of pancreatitis were observed in group C. Noteworthily, the concentrations of serum TNF-a and IL-lp in group A were higher than that in group B, the pathological changes were severer and the score of pathological changes of lung tissue in group A were higher significantly than that in group B (PO.01). The W/D ratio in group A was higher than that in group B (P<0.01), accordingly, the PaC>2 level in group A was lower than that in group B (P<0.01). Those results indicated that the inactivation of Kupffer cell did not change the severity of pancreatitis, but reduced significantly the level of serum inflammatory factors and alleviated remarkably the lung injuries. 2. The cultured hepatocytes had typical shape, well viability and a... |
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