An Improved Acute Pancreatitis Apoptosis/Necrosis In vitroModel of Acinar Cells

Abstract

To improve the apoptosis/necrosis In vitromodel of ascites-induced acute pancreatitic (AP) acinar cells by using the co-culture system and mixed ascites technology for the first time. Furthermore, we compared this improved model with cerulein and cerulein+LPS models, and observed the effects of three models on acinar apoptotic/necrotic-related indicators. Methods The In vitrocultured rat acinar cells AR42J were divided in four groups: control group (medium+PBS), cerulein group (medium+10 nmol/L cerulein), cerulein+LPS group (medium+10 nmol/L cerulein+10 mg/L LPS) and improved ascites group. In the improved ascites group, the ascites of sodium taurocholate-induced rat model was mixed and added into the co-culture system to stimulated In vitrocultured homogenous acinar cells. The co-culture system was set as follows: the chambers with the pore size of 1 μm were placed in the cultue plate, and the culture medium and mixed ascites were respectively added to the culture plate and the chamber at a ratio of 1∶1. The acinar cells in each group were collected after 24 h stimulation. The apoptotic/necrotic rates, the expressions of apoptosis/necrosis related proteins 〔B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X protein (Bax) and receptor interacting protein 1 (RIP1)〕, and the ultra-structure of acinar cells were detected by flow cytometry, Western blot and transmission electron microscopy (TEM). Results The acinar cells in the improved ascites model were mainly characterized by necrosis; compared with the other 3 groups, the apoptosis rate and necrosis rate were both up-regulated, RIP1 and BAX protein expression levels were up-regulated, and Bcl-2 protein was down-regulated. TEM results showed the organelle structure of acinar cells was destroyed, and the cell membrane was degraded in the improved ascites model. Compared with the control group, apoptosis rate of acinar cells in the cerulein and cerulein+LPS models were increased and necrosis rate were not changed. The expression of pro-apoptotic protein Bax was increased, while the expression level of RIP1 was not significantly increased. TEM results showed that in cerulein group and cerulein+LPS group,the chromatin of the cells was condensed into a mass, the cytoplasm was degraded and the cell membrane was intact, showing typical apoptosis characteristics. Conclusion Compared with cerulein and cerulein +LPS models, which mainly focus on apoptosis of acinar cells and applied to mild acute pancreatitis study, the improved ascites model mainly focuses on the necrosis of acinar cells and is a good model for studying acinar cell necrosis and severe acute pancreatitis.

Keywords: Acute pancreatitis, In vitromodel, Apoptosis/necrosis, Ascites