DKK-1调控组蛋白H3K27三甲基化在脂肪干细胞修复放射性肺纤维化过程中的作用机制研究

Ionizing irradiation stress-induced robust activation of Wnt signalling pathway plays a critical role in epithelial-mesenchymal transition (EMT) in irradiated type II alveolar epithelial cells and in proliferation of pulmonary fibroblasts, which attribute to fibrotic lesion formation in irradiated lungs. Our preliminary results indicated that pathogenesis of radiation-induced pulmonary fibrosis could be restricted by systemic delivery of human adipose-derived stem cells(ADSCs), presenting by the inhibitory effects of ADSCs on EMT in irradiated type II alveolar epithelial cells as well as tri-methylation in histone3 lysine 27 (H3K27me3) confered by DKK-1, a Wnt antagonist. However, the action by which DKK-1 mediated blockade of Wnt signalling pathway will increase H3K27me3 in irradiated cells remains unknown. To address such a issue, we intend to block or activate Wnt signalling pathway though up-regulating or down-regulating DKK-1 expressions in ADSCs for exploring whether H3K27me3 is dependent on blockade of Wnt signalling pathway, and whether H3K27me3 paticipates in silencing gene expressions related to pulmonary fibrosis. This project aims to investigate the inhibitory effects of ADSCs on radiation-induced pulmonary fibrosis and the underlying molecular events upon the increase in H3K27me3 driven by DKK-1 in controlling pathogenesis of pulmonary fibrosis. Our results will provide new evidences for preventing or treating radiation-induced pulmonary fibrosis clinically.

Wnt通路异常激活所介导的II型肺泡上皮细胞的上皮-间质转分化及肺固有成纤维细胞的增殖是引起放射性肺纤维化的重要因素。本课题组研究表明，静脉移植脂肪干细胞可抑制小鼠放射性肺纤维化的进展。其中，脂肪干细胞释放的Wnt通路拮抗剂DKK-1可抑制受照II型肺泡上皮细胞的上皮-间质转分化；并且DKK-1可上调受照II型肺泡上皮细胞H3K27me3的水平。但我们尚不清楚受照细胞H3K27me3水平的增高是否与DKK-1介导的Wnt通路受阻有关。对此，本项目拟通过上调/下调脂肪干细胞中DKK-1的表达来分别阻断/激活Wnt通路的信号转导，研究H3K27me3的水平与Wnt信号通路之间的关系，及其是否影响调控肺纤维化的关键转录因子的表达。旨在从表观遗传学层面探索脂肪干细胞介导的组蛋白H3K27me3水平的增高对放射性肺纤维化进展的抑制作用及相关分子机制。相关结果为放射性肺纤维化的防治提供新思路。

胸部放射治疗后发生肺损伤是限制放射治疗疗效的主要因素。电离辐射诱导的Wnt信号通路激活在II型肺泡上皮细胞的EMT和肺成纤维细胞的增殖激活中发挥了关键作用。人脂肪源性间充质干细胞Ad-MSCs 可以分泌Wnt通路拮抗剂DKK-1，其表达不受干细胞代次影响；Ad-MSCs来源的上清在体外能有效逆转电离辐射诱导上皮细胞E-cadherin表达的下降和Vimentin表达的增加，并抑制成纤维细胞增殖介质α-SMA的表达，从而抑制了EMT和成纤维细胞的激活；Ad-MSCs上清液有效减少了β-catenin入核，同时对EMT下游基因Snail, Twist、c-Myc也有抑制作用；此外，中和上清液中的DKK-1后，上述作用均发生逆转；体内研究证实，Ad-MSCs细胞尾静脉注射后，能有效升高血清中免疫抑制因子IL-10水平。综上所述，Ad-MSCs利用分泌的DKK-1，通过Wnt/β-catenin信号通路抑制肺泡上皮细胞EMT的发生，从而修复放射性肺纤维化。同时，在电离辐射引起诸多生物学效应中，DNA 双链断裂 （DSB）是最严重的一种损伤类型。在正常的肺组织中，DSB 的修复与放射性肺损伤的发生及严重程度密切相关。本项目研究发现，Ad-MSCs-exo 介导的旁分泌效应对 DSB 具有明确的修复作用。电离辐射可以诱导Ad-MSCs-exo 上调 miR-1246，靶向 LIG4，促进 NHEJ 通路参与 DNA 双链断裂的修复，使 细胞在接受电离辐射后 DSB 位点得以迅速被修复，减低辐射损伤。