胰蛋白酶原活化在胰腺炎发生中的机制研究

Premature trypsinogen activation has long been considered the key event in pancreatitis initiation. However, recent studies have questioned this conclusion, with new mouse models suggesting that trypsinogen activation is essential for the disease. It is reported that endoplasmic reticulum stress was early events in pancreatitis. We proposes the following assumptions in this study: trypsinogen activation increases severity of pancreas acinar cell apoptosis induced by endoplasmic reticulum stress. Also endoplasmic reticulum stress mediated positive feedback loop amplifies trypsin activity to pathological levels in pancreatitis. In the current study, two novel mouse models over-expressing human trypsinogen were examined to further elucidate the role of trypsin in acute and chronic pancreatitis. Transgenic mice were developed that over-expressed either wild-type or mutant human trypsinogens. Mice were examined under basal conditions and after caerulein induction of acute and chronic pancreatitis. Littermates not expressing the transgenes with same treatments were used as controls. Application of adenovirus overexpression of endoplasmic reticulum stress related genes and specific inhibitors. Using RT-PCR、Western blot、Immunohistochemistry and nuclear localization observed in pancreatic tissue and acinar cell to measure trypsin activity, pancreatitis parameters、inflammatory responses and ER Stress co-localization. Aimed at in-depth understanding of the role of trypsinogen activation in the pathogenesis of pancreatitis and reveal the mechanisms of their intracellular singling pathways and may be important strategy for pancreatitis.

胰蛋白酶原的活化在胰腺炎发生中的作用目前存在一定的争议，是否起始急、慢性胰腺炎及遗传性胰腺炎的发生尚未清楚。报道称内质网应激是胰腺炎发生的早期事件。本研究提出如下假设：胰蛋白酶原活化加重内质网应激引起的胰腺腺泡的凋亡，内质网应激能正反馈促使胰蛋白酶进一步活化促进胰腺炎的发生。本项目拟以胰腺腺泡细胞特异表达人野生型胰蛋白酶原和突变型胰蛋白酶原转基因小鼠为研究对象，以雨蛙素诱导的方法建立急、慢性胰腺炎动物模型，应用腺病毒过表达内质网应激相关基因，并应用特异性内质网应激抑制剂，采用RT-PCR、Western Blot、免疫组化、核定位等技术，观察胰腺组织和腺泡细胞胰蛋白酶原活性、细胞损伤和炎症反应、胰蛋白酶原与内质网应激相关基因共定位。旨在深入认识胰蛋白酶原活化在胰腺炎发病中的作用，并揭示其细胞内信号通路机制，阐明胰腺炎的发病机制，为探索胰腺炎的防治方法，提供新的切入点。

胰蛋白酶原的活化在胰腺炎发生中的作用目前仍然存在争议。有报道称内质网应激是胰腺炎发生的早期事件。本研究在此基础上提出胰蛋白酶原活化加重内质网应激引起的胰腺腺泡凋亡的假设，内质网应激能够正反馈促使胰蛋白酶进一步活化促进胰腺炎的发生。本项目以腺泡细胞特异表达人野生型胰蛋白酶和突变型胰蛋白酶原转基因小鼠为研究对象，以雨蛙素诱导的方法建立急慢性胰腺炎模型，采用腺病毒过表达相关基因和特异性内质网应激通路相关抑制剂干扰基因表达，结合RT-PCR、Western Blot、免疫组化、核定位等技术，观察胰腺组织和腺泡细胞的胰蛋白酶原活性、细胞损伤和炎症反应、胰蛋白酶原与内质网应激相关基因共定位。应用腺病毒成功构建并稳定繁育胰蛋白酶原转基因小鼠模型，并且验证了胰蛋白酶原活化转基因小鼠未干预无表型。通过雨蛙素腹腔注射法成功构建急性、慢性胰腺炎小鼠模型，结果证明胰蛋白酶原活化转基因组胰腺炎炎症明显比对照组严重，内质网应激通路的关键基因如CHOP、磷酸化-IRE1-a均显著高于对照组，提示内质网应激参与胰蛋白酶原活化相关的炎症过程。本研究为进一步认识胰蛋白酶原的活化在胰腺炎发病机制及细胞内信号通路机制提供了更多的科学依据，为探索胰腺炎的临床治疗方法提供新的切入点。