Chen Ke-Ling, Chen Jia-le, Chi Jun-Lin, Zhou Zong-Guang, Li Yuan
Institute of Digestive Surgery, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Department of Gastroenterological Surgery, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2018 Sep;49(5):765-770.
To improve the apoptosis/necrosis model 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.
The cultured 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 cultured 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).
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.
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.
首次利用共培养系统和混合腹水技术改进腹水诱导的急性胰腺炎（AP）腺泡细胞凋亡/坏死模型。此外，我们将这种改进后的模型与雨蛙肽和雨蛙肽+脂多糖模型进行比较，观察三种模型对腺泡凋亡/坏死相关指标的影响。
将培养的大鼠腺泡细胞AR42J分为四组：对照组（培养基+磷酸盐缓冲液）、雨蛙肽组（培养基+10 nmol/L雨蛙肽）、雨蛙肽+脂多糖组（培养基+10 nmol/L雨蛙肽+10 mg/L脂多糖）和改进腹水组。在改进腹水组中，将牛磺胆酸钠诱导的大鼠模型腹水混合后加入共培养系统，以刺激培养的同源腺泡细胞。共培养系统设置如下：将孔径为1μm的小室置于培养板中，培养基和混合腹水分别以1:1的比例加入培养板和小室。刺激24小时后收集每组的腺泡细胞。通过流式细胞术、蛋白质免疫印迹法和透射电子显微镜（TEM）检测凋亡/坏死率、凋亡/坏死相关蛋白[B细胞淋巴瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）和受体相互作用蛋白1（RIP1）]的表达以及腺泡细胞的超微结构。
改进腹水模型中的腺泡细胞主要表现为坏死；与其他3组相比，凋亡率和坏死率均上调，RIP1和BAX蛋白表达水平上调，Bcl-2蛋白下调。TEM结果显示，改进腹水模型中腺泡细胞的细胞器结构被破坏，细胞膜降解。与对照组相比，雨蛙肽和雨蛙肽+脂多糖模型中腺泡细胞的凋亡率增加，坏死率未改变。促凋亡蛋白Bax的表达增加，而RIP1的表达水平未显著增加。TEM结果显示，在雨蛙肽组和雨蛙肽+脂多糖组中，细胞染色质浓缩成块，细胞质降解，细胞膜完整，呈现典型的凋亡特征。
与主要关注腺泡细胞凋亡并应用于轻度急性胰腺炎研究的雨蛙肽和雨蛙肽+脂多糖模型相比，改进后的腹水模型主要关注腺泡细胞坏死，是研究腺泡细胞坏死和重症急性胰腺炎的良好模型。
