以整合素αv亚基为靶点干预治疗放射性肺损伤的作用机制及可行性研究

The radiation induced pulmonary fibrosis (RIPF) has high incidence and mortality rate without effective treatment. However, the underlying mechanism remains unclear. TGFβ1 has been intensely investigated in the pathogenesis of RIPF in recent years. And it is considered as a key player during RIPF. During the early stage, the latent-TGFβ is activated by αv integrins, which recognize and bind to RGD motif (arginine-glycine-aspartic acid sequence) within latency associated peptide (LAP). The activation of TGFβ is the beginning of RIPF, then the activated TGFβ may trigger downstream effects, including cytokine storm, through Smad and non-Smad singnaling. Our early study has found the single nucleotide polymorphism at rs1982073 of the TGFβ1 gene is associated with the risk of RIPF in patients with non-small-cell lung cancer treated with definitive radiotherapy and may serve as a reliable predictor of RP. Moreover, DNA array analysis showed that there is extensive crosstalk between αv integrins and TGF-β signaling during epithelial-mesenchymal transition (EMT) and radiation induced fibrosis. Interestingly, expression of interleukin-8 is up-regulated during the process. Synthesized RGD peptide can attenuate radiation-induced EMT and fibrosis by blocking the interaction between αv integrins and TGFβ. According to these findings, we assume that αv subunit is core element of αv integrins, which play critical role in RIPF. Furthermore, "αv integrins-TGFβ-inteleukin-8" is a positive feedback loop in radiation injury. Targeting and inhibiting αv subunit may reduce radiation-induced EMT and fibrosis. Inhibition of αv is a promising new approach for antifibrosis therapy. This study will investigate the function and role of αv subunit and other β subunits in RIPF, especially by conditional knock-out mice model. DNA Chip and tissue micro-array will be taken to look into the positive feedback loop mentioned above, identifying key protein in the regulation network. The aim of this study is to reveal the precise molecular mechanism of αv subunit in regulation of RIPF. Our study may provide new insight into RIPF and subsequently allows development of a novel targeted therapy strategies.

放射性肺纤维化（RIPF）是肺癌患者接受治疗后最常见并发症，目前仍缺乏有效防治手段。申请者前期研究发现TGFβ1 rs1982073不同基因型与RIPF发生密切相关；基因芯片分析证实人胚肺纤维母细胞经放射处理后不同整合素αvβ亚型与TGFβ1之间存在交叉对话，均伴随IL-8表达上调；利用RGD多肽干扰整合素与TGFβ1前体结合，可降低整合素αvβ3的表达并使放射诱导"上皮-间质转化"的进程受阻。申请人假设αv亚基作为整合素αvβ的核心组分，是激活TGFβ1诱导RIPF的关键，在RIPF发生中存在"整合素αvβ-TGFβ1-IL-8"正反馈调节，以αv亚基作为靶点进行干预可减轻RIPF的形成。本研究主要利用基因敲低及敲除技术筛选鉴定在RIPF中发挥关键作用的整合素亚基，探究上述正反馈通路调节机制，进而揭示其导致RIPF的分子机制，为寻找更有效的分子靶点提供理论依据。

放射性肺损伤是胸部放射治疗的常见并发症，极大限制了放射治疗的剂量。然而，目前尚无有效的方式防治放射性肺损伤。研究表明，靶向肌成纤维细胞整合素αv可减轻肝纤维化和心肌纤维化，但尚无研究证明整合素αv和放射性肺损伤的相关性。本研究利用慢病毒转染技术，构建了过表达整合素αv和整合素αv敲低的成纤维细胞系，检测了放射线诱导后各组细胞整合素αv和纤维化指标的表达差异。成功构建了ItgavloxP/loxP;Pdgfrb-Cre转基因小鼠并建立放射性肺纤维化动物模型，在动物和细胞水平研究了靶向整合素αv干预放射性肺纤维化的可行性。本课题研究了整合素基因单核苷酸多态性与放射性肺损伤发病的相关性。本项目研究表明：整合素α在放射性肺纤维化中具有重要作用，放射线诱导和（或）慢病毒感染干预成纤维细胞后，各组细胞纤维化指标蛋白的表达具有显著差异；敲除小鼠肌成纤维细胞整合素αv能显著减轻放射性肺纤维化程度。在前期研究基础上，我们发现在接受放疗的肺癌患者中，ITGB6基因rs4665162的AG/GG基因型患者肺组织表达较高的整合素蛋白，与放射性肺损伤发生风险较高显著相关。本课题的研究结果为临床上以整合素αv为靶点干预放射肺纤维化提供了理论依据。