TGF-β与Hedgehog通路互话（Crosstalk）参与胰腺星状细胞上皮间质转化（EMT）介导的胰腺纤维化相关机制研究

Activation of pancreatic stellate cells (PSCs) and subsequent interstitial fibrosis are considered as critical step in the pathogenesis of chronic pancreatitis. TGF-β/Smad is one of the major activating pathway of PSCs, however, its downstream molecular mechanisms are not fully understood. Our preliminary experiments have shown that transcription factor Gli in hedgehog signaling palyed a role in TGF-β1-stimulated PSCs activation. Based on this new findings and together with our experiment results from studies on epithelial-mesenchymal transition in PSCs, we proposed a new hypothesis: crosstalk between TGF-β and hedgehog took place on the level of transcription factor Gli, and promote the activation of PSCs via its pro-EMT ability. To validate our hypothesis, pathological observation and in vitro experiments were carried out. RNAi technology or pathway inhibitors were utilized to block TGF-β/hedgehog signaling or EMT process, and their effects on PSCs activation were evaluated. In our research project, crosstalk between TGF-β and hedgehog signaling pathway and its role in PSCs activation were validated, and EMT of PSCs as a possible mediating process in PSCs activation was also studied. We aim to clarify the mechanism in pathogenesis of pancreatic fibrosis, and to provide new targets for lab research and clinical intervention for chronic pancreatitis.

胰腺星状细胞（PSCs）激活所致的间质纤维化是慢性胰腺炎发病机制中的关键事件。TGF-β/Smad是PSCs激活的重要通路，但其下游分子机制尚不明确。我们的预实验提示：TGF-β1促PSCs激活过程需要Hedgehog通路转录因子Gli的参与。结合所开展的PSCs上皮间质转化（EMT）的相关研究结果，我们提出假说：TGF-β与Hedgehog通路在转录因子Gli环节发生互话（Crosstalk），通过调控细胞EMT，促进PSCs激活。为了验证这一假说，我们将开展临床病理学观察，并通过抑制剂或RNAi技术进行干预实验，干扰PSCs内TGF-β和Hedgehog通路及EMT过程，观察其对于细胞激活过程的影响。本研究将从TGF-β与Hedgehog通路发生互话，并调控PSCs发生依赖于EMT的激活这个新角度，揭示胰腺纤维化发生发展新的机制，为治疗慢性胰腺炎提供新的研究方向和治疗靶点。

胰腺纤维化是胰腺疾病重要的间质表现，包括在慢性胰腺炎中的主要病理改变，以及胰腺癌癌巢周围的结缔组织反应。目前我们已知，胰腺星状细胞（Pancreatic stellate cells, PSCs）激活是胰腺纤维化的关键核心事件。既往研究表明，TGF-β以及Hedgehog信号通路是PSCs激活的重要通路，但其激活的具体分子调控机制尚不明确，而TGF-β以及Hedgehog信号通路在其中的作用和地位也不明确。.本课题的第一部分重点着眼于PSCs在体外培养条件下的激活过程，开展了细胞功能实验，并检测了EMT相关基因表达。我们发现伴随着PSCs激活，细胞迁移能力显著增强。在mRNA以及蛋白质水平，相关基因表达量的改变提示EMT机制参与了PSCs激活过程：上皮表型基因表达降低，间质表型基因表达升高，同时EMT关键转录因子Snail和Slug的表达亦显著升高。本部分的研究结果对于探索PSCs发生来源，以及对于PSCs激活过程调控机制的深入研究具有重要意义。.在第二部分实验中，我们发现TGF-β和Hedgehog信号通路在胰腺纤维化中均显著激活，而Hedgehog信号通路转录因子Gli阻断剂，而非其上游分子阻断剂能够阻断外源性TGF-β1对于胰腺星状细胞的激活作用。同时分别通过检测下游基因表达以及双荧光素酶报告质粒系统两种方法，我们证实TGF-β1能够促进Hedgehog信号通路的激活。结合生物信息学手段，我们推测TGF- β可能通过诱导Hedgehog信号通路转录因子Gli的表达增加来活化Hedgehog信号通路，我们进一步通过Smad2/3的siRNA干扰实验，证明TGF- β1主要通过Smad2/3来实现对于Gli1和Gli2的转录水平的正性调控。