维甲酸诱导胰腺星状细胞分化及其逆转胰腺纤维化的机制研究

Chronic pancreatitis (CP) is characterized by progressive pancreatic fibrosis.It was well established that the persistent activation of pancreatic stellate cells (PSCs) plays a pivotal role in the pancreatic fibrogenesis, while the differentiation and outcome of activated PSC determines the pathological outcome after pancreatic injury. Previously, we found that retinoic acid receptors and SFRP were markedly downregulated during pancreatic fibrogenesis, while β-catenin was upregulated. Furthermore, retinoic acid can inhibit the CP of pancreatic fibrosis induced by TNBS in rats. Thus, we speculate that in the CP process, retinoic acid signaling pathway in the quiescence of the PSC from the autocrine / paracrine SFRP inhibition of the Wnt signaling pathway is blocked, leading to dynamic imbalance of PSC activation and differentiation or outcome, thereby causing the pancreas fibrosis. The purpose of this study is to investigate the potential effects of retinoic acid on the differentiation and outcome of PSCs through both in vitro and in vivo experiments, thereby clarifying the molecular mechanism of retinoic acid/retinoic acid receptor pathway in the pancreatic fibrogenesis. Specifically, we propose to conduct the following works: ① Using in vitro cell-based systems, to observe the changes in the expression of retinoic acid receptor, SFRP and Wnt signaling molecules and to determine its relationship with pancreatic fibrosis; ② On the animal level (knockout mice), to investigate the effects of retinoic acid on PSC activation, differentiation and outcome; ③ To investigate the effects of retinoic acid on the retinoic acid receptor, SFRP and Wnt signaling pathway of PSC, to clarify the retinoic acid signaling pathway to regulate the molecular mechanisms of pancreatic fibrosis. These results will help unravel the molecular pathogenesis of mechanism of CP and may provide insight into the development of novel therapeutic agent for CP.

慢性胰腺炎（CP）以胰腺纤维化为主要病理特征，而胰腺星状细胞（PSC）持续活化是胰腺纤维化的核心机制，PSC的分化和转归决定了胰腺损伤后的病理结局。我们前期发现，在PSC的活化过程中存在维甲酸受体、SFRP下调和β-catenin的上调，同时维甲酸能抑制TNBS诱导的大鼠CP的胰腺纤维化。因此，我们推测在CP的发生过程中，维甲酸信号通路存在抑制，PSC通过自分泌/旁分泌SFRP抑制Wnt信号通路被阻断，导致PSC活化和分化、转归的动态失衡，进而引起胰腺纤维化。本研究拟：①建立细胞培养为主的体外模型，运用分子细胞生物学方法，观察维甲酸受体、SFRP和Wnt信号分子的表达变化，确定其与胰腺纤维化的关系；②建立基因敲除小鼠体内模型，观察维甲酸信号通路对PSC活化和转归以及胰腺纤维化的影响；③研究维甲酸对PSC中SFRP/Wnt信号通路的影响，及其调节胰腺纤维化的分子机制，并探讨其潜在的临床价值。

胰腺纤维化是慢性胰腺炎（CP）的主要病理特征，可造成胰腺组织和功能的持续性和永久性损害。当胰腺受损或有炎症时，胰腺星状细胞（PSC）是细胞外基质（ECM）沉积的主要来源，且PSC的持续活化是胰腺纤维化进展的核心机制，PSC的分化和转归决定了胰腺损伤后的病理结局。近来来PSC持续活化在CP纤维化中的作用也受到越来越多的关注。.Wnt蛋白是包括19个家族在内的糖蛋白，不仅具有高度保守性且能分泌丰富的半胱氨酸，它参与调控细胞的生长、增殖、迁移、分化、癌变、凋亡及干细胞自我更新等过程。。有研究表明，Wnt信号通路与多种器官纤维化的发生有关，特别是与肝脏纤维化有密切关系。而且我们团队在之前的研究中已经发现：Wnt/β-catenin信号通路可以通过上调Wnt 2和β-catenin的蛋白水平来调控PSC的活化和增加核β-catenin的表达。维甲酸（RA），是一类维生素A，它有广泛的生物学效应，包括调节细胞的分化和增殖，以及减轻肝脏、心脏、肺脏和肾脏等重要器官的纤维化。然而，RA调控Wnt信号通路与胰腺纤维化关系的机制却仍不清楚。本课题试图研究RA通过调控Wnt/β-catenin信号通路来观察对体内胰腺纤维化及体外活化PSC的作用。.本课题通过在小鼠腹腔内有规律地注射雨蛙肽来诱导反复发作性的胰腺炎，从而建立CP模型，并应用小鼠胰腺组织进行酶消化法和Nycodenz密度梯度离心法分离原代PSC。由实验结果发现：RA能通过降低胰腺组织的损害，减少体内α-SMA的表达及下调β-catenin、TGFβRII、PDGFR和collagen 1α1的mRNA水平来抑制胰腺的纤维化。在经RA处理后，体内和体外的Wnt2和β-catenin的蛋白表达水平明显下调，而Axin 2的表达水平上调。且我们的研究结果还表明：与小鼠CP和活化的PSC相比，RA抑制了β-catenin的核转位。除此之外，RA能明显抑制PSC的增殖和促进PSC的凋亡，且能通过下调β-catenin、TGFβRII、PDGFR和collagen 1α1的mRNA的表达来抑制细胞外基质的生成。以上一系列的实验结果都说明了维甲酸能够通过调控Wnt/β-catenin信号通路来抑制胰腺星状细胞的活化及逆转胰腺纤维化，且RA可作为一种高效的药物来治疗慢性胰腺炎。