TnC基因异构体TnC-FNⅢD在百草枯中毒肺纤维化中的作用及相关机制研究

Paraquat (PQ) poisoning is the most lethal pesticide suicide and the main cause of death is lung fibrosis. Up to date, the mechanism underlying PQ poisoning has not been completely understood and there is no effective antagonist. ATⅡcells play an important role in the process of lung fibrosis induced by PQ. In previous work, we found that tenascin C (TnC ) expression of human ATⅡcells increased when exposed to PQ. And in mice model of PQ poisoning, TnC splice isoforms- FNⅢD increased significantly in lung tissues. And the elevation has positive coralation with lung fibrosis severity. So we speculate that ATⅡcells overexpress TnC when stimilated by inflammation mediators induced by PQ poisoning and produce many TnC splice isoforms-FNⅢD under some unknown regulatory factors, which directly take part in lung fibrosis. The function and regulatory network of FNⅢD gene will be well studied in this project. We aim to study the biological characteristics of FNⅢD gene and clarify its position in cellular signaling pathway by molecular cytogenetic and bioinformatics tools and transgenic mouse, together with the therapeutic effect of RNAi FNⅢD gene. This project will provide valuable data for clarifying the molecular mechanism of lung fibrosis in PQ poisoning. In clinical terms, we hope to find new ideas for diagnosis and molecular-targeted therapy in PQ poisoning lung fibrosis.

百草枯(PQ) 中毒是目前致死性最高的农药中毒事件，中毒后引起的肺间质纤维化是导致死亡的主要原因。ATⅡ细胞在PQ中毒导致的肺纤维化过程中扮演重要角色，申请者在完成上一个国家自然基金的过程中发现，PQ中毒小鼠肺组织中TnC拼接异构体-FNⅢD在PQ中毒小鼠肺组织中明显升高，且与肺纤维化程度明显正相关。因此我们推测在PQ中毒过程中，ATⅡ细胞在大量氧自由基刺激下过度表达TnC且在某些未知调控因子的作用下形成大量TnC拼接异构体FNⅢD，直接参与肺纤维化的形成，FNⅢD基因可能是PQ中毒肺纤维化的重要靶点,国内外尚无相关研究报道。本研究利用分子细胞遗传学、生物信息学手段并构建转基因小鼠，探索该FNⅢD基因在PQ中毒肺纤维化中的功能和启动子调控机制，寻找其下游调控蛋白，并研究RNAi干扰FNⅢD基因的治疗作用，为PQ中毒肺纤维化发生的分子机制及临床诊治提供有价值的研究资料和治疗靶点。

本研究首先明确了TNC在PQ中毒小鼠肺脏中以及在PQ刺激的ATII细胞中的表达情况，并发现TNC是链接PQ中毒后急性肺损伤和肺纤维化的关键调控蛋白。TNC在PQ中毒急性肺损伤和肺纤维化过程中表达明显升高，且和α-SMA、NF-κB p65、TGF-β1表达和肺纤维化评分明显正相关，提示TNC是连接肺脏炎性损伤和肺纤维化的重要介质。 TNC与TGF-β1之间存在着相互促进作用，TNC贯穿PQ诱导的肺损伤的炎症和纤维化两个阶段。 TNC基因敲除后，通过降低TLR4和TGFβ1的表达，既减轻了PQ所致的急性肺损伤，又阻断了后续的肺纤维化过程，提示TNC可能是临床干预的一个理想的靶点。临床研究发现，TNC是急性PQ中毒患者预后不良的血清标志物，而且和肺损伤程度正相关，填补了临床缺乏预后评估标志物的空白。iTRAQ+PRM结果提示多条信号通路在PQ中毒小鼠肺损伤中发挥重要作用，免疫相关信号通路的不同时间点表达的特点，提示PQ中毒肺损伤可能存在自身免疫系统过度活化的二次打击学说，从分子水平揭示了PQ肺损伤的相关机制。巨噬细胞活化在PQ中毒肺损伤中发挥重要作用，初步研究发现通过调节巨噬细胞的糖代谢通路，可以降低巨噬细胞M1极化，有望从免疫调节角度减轻PQ中毒肺损伤程度。这也是后续研究的重点。.