大蹼铃蟾三叶因子（Bm-TFF2）在对其自身皮肤创伤修复中的作用及其机制研究

The signal transduction and regulation of amphibian skin regeneration is an very important and interesting issue in scientific world. We focus on the study of the skin of Chinese belly toad (Bombina maxima) and presented the fundamental data of some bioactive components, including the secreted proteins and peptides of the skin which is pivotal for deserting its physiological funtion. A trefoil factor (TFF), Bm-TFF2, has been purified from the B.maxima skin and is able to bind and activate protease activated receptor 1 (PAR1) to induce platelet activation. Our present innovative results show that Bm-TFF2 could induce epthelial cell migration and wound healing of human gastric and intestina carcinoma by activated both PAR1 and PAR2. We found the relationship between human trefoil factor 2 (hTFF2) and PARs and the new mechnisms that regulated PARs activation for the first time.. Human TFFs participated in various crucial biological process and play an important role in wound healing, epithelial restituteion and anti-inflammatory effects in the gastrointestinal tract by stimulating cell migration and inhibiting cell apoptosis. However, its receptors, signaling pathways and regulative mechanisms are still not known over the past several decades. Besides the function of Bm-TFF2 on epethelial cell migration and wound healing is consistent with that of human TFFs, we also noticed that the concentrations of Bm-TFF2 used were much lower. hTFFs are highly expressed in gastrointestinal tract, but had relatively weak activities. So, it is hard to investigate the roles and regulative machinisms of hTFFs. In comparison with hTFFs, Bm-TFF2 is more potent in the induction of cell migration and wound healing. Bm-TFF2 should be a useful molecular tools for basic research on the pathophysiological roles of TFFs proteins.. Using mammalian cell could not comprehensively intepret the roles and functional mechanisms of Bm-TFF2, The aims of our project are to: (1) anaylize the expression and distribution of Bm-TFF2 in the skin of B.maxima during the process of skin injury and wound healing. 2）clarify the relationship between the signaling pathway of PARs receptor activated by Bm-TFF2 and the skin injury and wound healing of B.maxima from both cell and tissue level. This research in this project will investigate the function of Bm-TFF2 in B.maxima skin injury and wound healing and aid new sight into the functions and molecular mechanisms of mammalian TFFs and skin regeneration.

以大蹼铃蟾为研究对象，从其皮肤分泌物中得到两栖类三叶因子Bm-TFF2；前期工作表明Bm-TFF2通过激活人蛋白酶激活受体PAR1诱导人血小板聚集，共同激活PAR1和PAR2促进人胃癌和肠癌上皮细胞迁移。首次揭示人三叶因子2（hTFF2）与PAR受体之间的关联并发现了PAR1激活调控的新机制。为体内分布广泛、生物学作用明确但具体机制未明的哺乳动物TFF的研究提供了一个崭新的视角。但以哺乳动物细胞作为研究体系，不能回答Bm-TFF2在皮肤上的生物学功能。本项目的研究旨在:1)解析Bm-TFF2在大蹼铃蟾皮肤创伤修复过程中的表达及分布情况。2）在细胞和组织水平探讨依赖Bm-TFF2的PAR受体信号通路与大蹼铃蟾皮肤创伤修复的关系。本研究将阐明Bm-TFF2在两栖类动物皮肤创伤修复中的作用，研究成果可望为哺乳动物三叶因子的研究及皮肤再生的信号调控作用机制提供新的思路及方向。

本项目经过2013年-2015年三年的执行期，基本上按照计划进行并开展了计划书中所列出的主要内容。包括：1）大蹼铃蟾Bm-TFF2在皮肤和其它组织的表达分布。Bm-TFF2在皮肤创伤修复过程中的表达及分布情况。 2）Bm-TFF2在大蹼铃蟾皮肤中的生物学功能，包括对于大蹼铃蟾皮肤组织和表皮细胞以及成纤维细胞的生理活性测定，包括组织生长和坏死，细胞增殖、迁移和凋亡等。 3）探索Bm-TFF2对大蹼铃蟾PAR-PHB信号通路的作用，包括大蹼铃蟾PAR和PHB基因的克隆和表达，Bm-TFF2对大蹼铃蟾表皮细胞以及成纤维细胞中PAR-PHB信号通路的激活或抑制活性检测。原计划的前两项内容都已经完成，而第三项也已经完成了PAR和PHB的基因克隆和表达，相关信号通路的研究正在进行中。在保证原有研究计划的基础上，增加了人TFF、PAR和PHB相关的病理学研究，并取得了一些成果。发现肺癌组织中，PAR4的表达下调。也发现在胃癌组织中TFF2的表达下调是由于其启动子甲基化所致。这些研究结果虽然不是以两栖动物为研究对象开展，但是却为理解TFF-PAR-PHB信号通路的机制提供了基础，有利于后期的持续研究。在开展本项目的研究过程中，我们也发现了一些问题，包括：（1）两栖动物细胞传代次数受限，实验规模难以扩大；（2）两栖动物的冬眠期实验结果不稳定。然而在项目的探索中也积累了两栖动物细胞培养和整体动物饲养的经验，有利于后期实验的开展。