Ac-SDKP抑制矽肺肌成纤维细胞分化作用关键调节蛋白的筛选

Silicosis is one of the most severe occupational diseases in China at the present time, and its prevention and treatment have always been an important question in the occupational disease research. Recent studies indicate that the differentiation from mesenchymal cells and epithelial cells to myofibroblasts induced by TGF-β is an important new target in the study of molecular mechanisms of organ fibrosis (including lung fibrosis). For the first time, our research group reported the anti-silicotic effect of Ac-SDKP in rat silicosis model. Recently, we have found that Ac-SDKP could exert its anti-fibrotic effect by inhibiting the differentiation of myofibroblasts in vivo and in vitro, but the specific mechanisms are still not clear. We are going to use the in vitro experimental model in which TGF-β induces the differentiation from lung fibroblasts and type II alveolar epithelial cells to myofibrobalsts for this study, and then give the treatment of Ac-SDKP. We will analyze the dynamic expression of differential proteins in different time points and compare the proteins produced in the differentiation process from two different cells by using comparative proteomics. We will identify the specific proteins related to the therapeutic effect of Ac-SDKP. And we will answer the question whether or not Ac-SDKP has the same regulatory mechanism during the myofibroblasts differentiation from mesenchymal cells and epithelial cells. We will screen out and identify the key regulatory proteins related to silicosis and the anti-silicotic effect of Ac-SDKP through comprehensive analysis of the proteomics in silicotic model.

尘肺病（包括矽肺）是目前我国危害性最为严重的职业病，对其防治研究一直是职业病研究领域的重点课题。最近研究表明肌成纤维细胞的分化是矽肺纤维化发生机制与防治研究的重要靶标。课题组首次报道了Ac-SDKP具有防治大鼠矽肺纤维化的作用。最近又发现Ac-SDKP能够通过抑制肺肌成纤维细胞分化而发挥其抗矽肺纤维化作用，但其机制不甚清楚。本研究拟采用TGF-β1诱导刺激肺成纤维细胞和肺泡II型上皮细胞向肌成纤维细胞分化的体外模型，并给予Ac-SDKP干预。应用蛋白质组学技术，动态比较各时间点差异蛋白的表达，分析两种细胞在转化过程中差异蛋白的异同。发掘与Ac-SDKP干预作用相关的特异蛋白。揭示在两种细胞向肌成纤维细胞分化时，Ac-SDKP是否具有相同或不同调控作用的机制。结合矽肺模型蛋白质组学结果进行综合分析，筛选与挖掘出与矽肺发生相关的关键调节蛋白，以及与Ac-SDKP抗矽肺纤维化作用相关的调控靶点。

N-乙酰基-丝氨酰-天门冬氨酰-赖氨酰-脯氨酸（N-acetyl-seryl-aspartyl-lysyl-proline, Ac-SDKP）是一类由体内合成的，具有抑制炎症和纤维化（心、肺、肝、肾）形成的短肽，在其基础分泌水平，就有抑制胶原合成的作用。相反，其水平的降低能够诱导促纤维化因子的表达和细胞外基质沉积。课题组首次证明了Ac-SDKP能够在体内外抑制矽肺大鼠纤维化的发生、发展，与抑制细胞增殖、肌成纤维细胞分化和胶原合成有关。在本研究中，课题组采用双向凝胶电泳技术（two-dimensional gel electrophoresis, 2-DE）和同位素标记相对和绝对定量（isobaric tags for relative and absolute quantitation，iTRAQ）方法，分别筛选了大鼠原代肺成纤维细胞和A549肺泡II型上皮细胞向肌成纤维细胞分化的关键调节蛋白。基于蛋白组学结果，课题组还发现了乙酰化微管蛋白α （acetylated α-tubulin, Ac-α-Tub）在矽肺纤维化中缺失表达，而Ac-SDKP能够通过对血管紧张素（angiotensin, Ang）II和Rho相关卷曲螺旋蛋白激酶（Rho-associated coiled-coil kinase, ROCK）信号的阻断，抑制组蛋白去乙酰化酶（histone deacetylase, HDAC）6的上调，从而在体内外抑制肌成纤维细胞分化，稳定微管蛋白的表达，发挥了抗矽肺纤维化的作用。