TGFβ1-RGD模体与整合素αvβ3交互作用在放射性肺纤维化中的作用及机制研究

The Radiation Induced Pulmonary Fibrosis(RIPF) has high incidence and mortality rate,but has no effective treatment,because the pathogenesis remains unclear..TGFβ1 has been intensely investigated in the pathogenesis of RIPF in recent years.The TGFβ1 induced Epithelial-Mesenchymal Transition (EMT) may play a critical role in the pathogenesis of RIPF, while the TGFβ1 activation may be mediated by the integrinαvβ3 interacts with RGD motif (the arginine-glycine-aspartic acid sequence) located near the C terminus of TGFβ1..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 our follow study has demonstrated that cells transfected with differrent type of the TGFβ1 gene have both different EMT status and different expression level of integrinαvβ3. .Based on this research background, by means of cell transfection and TGF-β1 variant knock in mice , we design this project to investigate whether the RIPF is dependent on the EMT induced by TGFβ1 activation mediated by the integrinαvβ3 interacts with RGD motif,with three aspects:1)to investigate the cellular protein interaction ;2)to investigate the cell phenotype;3) to investigate the functional change in mouse model..In the in vitro experiment,we use immunoprecipitation and immunofluorescence method to investigate the interaction between TGFβ1 and αvβ3 in different cell lines which have been constructed by our study group transfected with differrent TGF-β1 genotype. Meanwhile ,we test both EMT in Alveolar Epithelial Cell lines and cellular differentiation in Human Embro Fibroblast cell lines induced by irradiation,the TGFβ1-Smad pathway protein and αvβ3 pathway protein expression level are also tested..In the in vivo experiment, Wild-type mice, TGF-β1 variant knock in mice(heterozygote and homozygote) are exposed to thoracic irradiation.Some mice are treated with RGDS peptide. Lung fibrosis are assessed by the pathologic grading or immunohistochemistry after irradiation. The expression level of TGFβ1,αvβ3 and the downstream signal pathway proteins are determined by immunohistochemistry,while the interaction between TGFβ1 and αvβ3 are also tested in the mouse's lung tissue protein. .Our studies are performed to demonstrate TGFβ1 activation mediated by the integrinαvβ3 interacts with RGD motif plays a key role in the pathogenesis of RIPF.Inhibition of αvβ3-mediated TGFβ1 activation is a promising new approach for antifibrosis therapy.

TGFβ1诱导的上皮间质转化(EMT) 是近年放射性肺纤维化(RIPF)机制研究新的动向。研究表明：TGFβ1与整合素αvβ3正反馈调节能促进EMT形成，且RGD模体是整合素αvβ3激活TGFβ1的关键位点。申请者前期人体研究发现TGFβ1rs1982073不同基因型与RIPF发生密切相关，且不同基因型细胞EMT形成及整合素αvβ3表达亦存在差异。据此本研究假设：TGFβ1-RGD模体与整合素αvβ3交互作用在RIPF形成中可能起关键作用，且是不同基因型导致RIPF发生差异的原因。本研究拟采用分子转染技术，构建TGFβ1rs1982073不同基因型细胞，研究TGFβ1与整合素αvβ3的交互作用及关键点，以及在RIPF形成中的功能及其下游信号分子变化；通过转基因技术构建TGFβ1rs1982073 变异型动物模型进行效应及功能研究验证本假设，以期找到干预RIPF的分子靶点，提高肺癌放疗疗效。

本研究利用慢病毒转染技术，构建了TGFβ1rs1982073不同基因型Ⅱ型肺泡上皮细胞，检测了放射线诱导和RGD多肽干预后各组细胞TGFβ1与整合素αvβ3表达差异以及各组细胞发生上皮间质转化(EMT)差异。成功构建了TGFβ1不同基因型转基因小鼠并建立放射性肺纤维化(RIPF)动物模型，在动物和细胞水平研究了RGD多肽干预放射性肺纤维化的可行性。本课题建立了含肺癌放疗患者420例的中国汉族人群放射性肺损伤的生物标本库，并完成随访310例。本项目研究表明：TGFβ1与整合素αvβ3交互作用在RIPF中具有重要作用。放射线诱导和RGD多肽干预TGFβ1不同基因型细胞后，各组细胞发生EMT具有显著差异，但与预期结果相反的是，这种现象以CC型更为明显；本研究在动物和细胞水平发现了RGD多肽能显著干预RIPF的发生，其中以低剂量组效果更为明显；我们根据前期基础研究筛选出的放射性肺损伤关键基因,发现了ITGB6，MMP1，Pi3k, PAI-1等多个基因中多个单核苷酸多态性位点对接受放疗肺癌患者中放射性肺损伤的发生具有预测作用。本课题的研究结果为临床上以TGFβ1与整合素αvβ3交互作用为靶点，利用RGD多肽干预放射肺损伤提供了理论依据。本课题发现了多个单核苷酸多态性位点对放射性肺损伤的发生具有预测作用，为放射性肺损伤的预测提供了新的标志物，为后期放射性肺损伤预测模型的建立提供了依据。