Trypsin-dependent mechanisms in pancreatitis

摘要 这项资助的主要目的是使用基因工程小鼠模型来确定 胰蛋白酶原（PRSS 1）突变在人类引起遗传性胰腺炎的机制。的 大多数慢性胰腺炎的非酒精性病例是在遗传易感性的基础上发展的， 由编码消化酶或其抑制剂的风险基因突变驱动，如阳离子 胰蛋白酶原（丝氨酸蛋白酶1，PRSS 1）、胰凝乳蛋白酶C（CTRC）或胰腺分泌性胰蛋白酶 抑制剂（丝氨酸蛋白酶抑制剂Kazal 1型，SPINK 1）。我们之前的研究确定了胰蛋白酶- 与这些风险基因突变相关的依赖性病理途径， 胰腺胰蛋白酶原自激活并导致异位胰蛋白酶活性增加。本 建议，我们将在体内验证这个模型。为此，我们开发了新的小鼠品系T7 D23 A和 在天然小鼠阳离子胰蛋白酶原（同种型）的活化肽中携带突变的T7 K24 R T7）。在体外，D23 A突变导致胰蛋白酶原自激活显著增加50倍，而 突变K24 R使自激活增加4倍。因此，模型可以提供关于如何 不同的胰蛋白酶水平决定胰腺炎反应和病理学。我们假设， 刺激自身激活的胰蛋白酶原突变将发展为自发性胰腺炎或表现出 当用过度刺激损伤进行挑战时，胰腺炎反应增强。在我们的具体目标中， 我们将研究T7 D23 A小鼠的自发性胰腺炎;评估对 促分泌素诱导的T7 K24 R小鼠胰腺炎的实验研究及胰蛋白酶的保护作用 通过改变这些细胞中Spink 3（人SPINK 1的直系同源物）表达水平来抑制胰腺炎 模型这些目标的成功完成将坚定地确立增加胰蛋白酶原 导致胰腺内胰蛋白酶活性升高的自身活化是胰腺炎的相关疾病机制。 胰腺炎，应该是未来治疗策略的重点。

ABSTRACT The main objective of this grant is to use genetically engineered mouse models to determine the mechanisms by which trypsinogen (PRSS1) mutations in humans cause hereditary pancreatitis. The majority of non-alcoholic cases of chronic pancreatitis develop on the basis of genetic susceptibility, driven by mutations in risk genes that encode digestive enzymes or their inhibitor, such as cationic trypsinogen (protease serine 1, PRSS1), chymotrypsin C (CTRC) or the pancreatic secretory trypsin inhibitor (serine protease inhibitor Kazal type 1, SPINK1). Our previous studies defined a trypsin- dependent pathological pathway associated with mutations in these risk genes that promote intra- pancreatic trypsinogen autoactivation and result in increased ectopic trypsin activity. In the present proposal, we will validate this model in vivo. To this end, we developed novel mouse lines T7 D23A and T7 K24R that carry mutations in the activation peptide of the native mouse cationic trypsinogen (isoform T7). In vitro the D23A mutation causes a dramatic 50-fold increase in trypsinogen autoactivation, while mutation K24R increases autoactivation by 4-fold. Thus, the models can provide information on how different trypsin levels determine pancreatitis responses and pathology. We hypothesize that mice with trypsinogen mutations that stimulate autoactivation will develop spontaneous pancreatitis or exhibit heightened pancreatitis responses when challenged with hyperstimulation insults. In our specific aims we will study the spontaneous pancreatitis in the T7 D23A mouse; evaluate the increased sensitivity to secretagogue induced pancreatitis in the T7 K24R mouse and investigate the protective role of trypsin inhibition against pancreatitis by altering Spink3 (ortholog of human SPINK1) expression levels in these models. Successful completion of these aims will firmly establish that increased trypsinogen autoactivation leading to elevated intra-pancreatic trypsin activity is a relevant disease-mechanism in pancreatitis and should be the focus of future therapeutic strategies.