NRP1调控细胞分化及放射性肺纤维化机制

Radiation therapy is currently one of the three major means of cancer treatment. About 60 to 70% of patients with malignant tumors are treated with radiation therapy during their treatment. In the radiotherapy of common clinical chest tumors (such as lung cancer, breast cancer, esophageal cancer, etc.), lung tissue as a moderately sensitive organ of radiation are damaged in varying degrees. Radiation induced lung injury, including early onset of radiation pneumonitis and subsequent radiation-induced pulmonary fibrosis (RIPF). The clinical manifestations are chronic respiratory failure and other irreversible symptoms, and the prognosis is very poor. Clinically, high dose corticosteroids are mainly used to treat the disease, but they do not effectively prevent the development of RILF. At present, the disease is still lack of specific and effective therapeutic drugs. Therefore, elucidating the pathogenesis of radiation-induced pulmonary fibrosis and looking for the key factors regulating cell differentiation in order to find an effective prevention and treatment of radiation-induced pulmonary fibrosis, radiotherapy for clinical chest tumors and prevent the development of RIPF drugs has important scientific significance..NRP1 (Neuropilin 1) is not only a surface marker of regulatory T cell (Treg) cells but also as a co-receptor for various growth factors, can enhances the activity and potential form of TGF-β1, and TGF-β1 is a main regulator of endothelial cell proliferation and migration, extracellular matrix metabolism, EMT and tumor metastasis. In previous studies, we have shown that NRP1 plays a regulatory role in the radiation-induced function and activation of Treg cells, and plays an important role in the radiation resistance of non-small cell lung cancer (NSCLC) cells. Our latest study found that EMT marker proteins (N-cadherin and Vimenrin) were downregulated and upregulated in irradiated lung epithelial models with NRP1 knockdown and high expression, respectively. In the mouse model of RIPF, the expression of NRP1 in lung tissue was higher at 4W, 8W, 16W and 24W after irradiation than that of the control group, and the expression level of CD25 protein in inflammatory phase (8W) was higher than that in pulmonary fibrosis (24W )..Based on the above preliminary studies, this project intends to use biological models of the mouse model of NRP1 knockout (NRP1-/-) and animal models of RIPF, as well as various cell models established in vitro, from both global and in vitro levels in order to explore NRP1 plays an important role in RIPF process and clarifies the cellular mechanism of NRP1 regulating Treg differentiation and Th1/2, Th17 cell balance and EMT related cell differentiation in different stages of RIPF. It further clarifies that NRP1 is involved in inflammatory stage and fibrosis of key signaling networks (TGF-β/Smads, Wnt/β-catenin and NF-κB pathway) and investigates the regulatory mechanisms of activation of downstream transcription factors (Twist, Snail and ZEB1). Providing the theoretical basis for the key regulatory factors, and then provide new ideas for the development of clinical precision medical solutions.

NRP1不仅是Treg细胞的表面标志，并且作为多种生长因子的辅助受体可增强TGF-β的活性，而TGF-β是调节内皮细胞增殖和迁移、上皮-间质转化、ECM代谢及肿瘤转移的主要调节剂。我们的先期研究发现，NRP1在辐射诱导的Treg细胞激活及其功能发挥中具有调控作用，同时在肺癌细胞辐射抗性中起重要作用，但NRP1在放射性肺纤维化（RIPF）进程中对各种细胞分化的调控规律及机制尚不清楚。本项目拟利用NRP1基因肺特异性敲除的小鼠（NRP1-/-）模型和RIPF动物疾病模型，以及体外建立的各种细胞模型等生物学模型，从整体水平和离体水平探讨NRP1在RIPF进程中的作用规律，阐明NRP1调控Treg细胞分化和Th1/2, Th17型细胞平衡，以及肺上皮细胞分化相关的细胞学机制，进一步明确NRP1参与RIPF进程中的关键信号网络调控机制，为寻找临床胸部放疗中出现RIPF的关键调控因子提供理论依据。

放射性肺纤维化（RIPF）作为常见的临床胸部恶性肿瘤放疗的并发症，尚缺乏特异、有效的治疗药物。因此，阐明RIPF的发病机制及寻找在其发生发展中调控细胞分化的关键因子，从而寻找RIPF有效防治的分子靶点，对临床胸部肿瘤的放疗和预防RIPF 药物的研发具有重要的科学意义。本项目利用NRP1基因肺特异性敲除的小鼠（NRP1-/-）模型和野生型小鼠疾病模型，以及体外建立的各种细胞模型阐明NRP1调控肺组织中的Treg细胞分化和Th1/2, Th17型细胞平衡，以及肺上皮细胞分化相关的细胞学机制，进一步明确NRP1参与RIPF进程中的关键信号网络调控机制，为寻找RIPF发生发展的关键靶点提供理论依据。.1..从离体水平和整体水平阐明了 NRP1 在RIPF发生过程中 Treg 细胞分化的细胞学机制以及炎症因子风暴，证实了在RIPF前期炎症阶段Treg细胞和各种Th细胞的分化是重要机制之一，从而阐明NRP1在RIPF发生发展中的重要作用。.2..以人和小鼠肺上皮细胞系为研究对象，在体外建立NRP1基因高、低表达的A549和MLE-12细胞模型，检测NRP1对辐射诱导的两种细胞上皮-间质转化（EMT）标志物表达变化及形态学变化，阐明了 NRP1 在RIPF发展中关键环节——发生EMT中的作用机制。同时，探讨了NRP1对辐射诱导A549细胞EMT相关转录因子（ZEB1，Twist）的影响，并分析了NRP1对辐射诱导A549和MLE-12细胞TGF-β1/Smads信号通路的影响。.3..采用野生型C57BL/6小鼠的RIPF模型，证明NRP1 在RIPF的发生发展中起到促进作用。之后，利用基因敲除小鼠模型反向证实NRP1对RIPF发生发展的影响。即建立并鉴定肺泡II型上皮细胞条件敲除NRP1小鼠模型，通过其肺组织基因表达谱测序，分析出差异表达基因以及生物学进程和KEGG数据。然后利用敲除NRP1小鼠建立RIPF模型并验证，进一步探讨在RIPF发生发展过程中NRP1对Treg细胞分化、各种细胞因子分泌、各种信号网络的影响以及对RIPF发展的抑制作用，在整体水平阐明了NRP1对RIPF发生发展的影响。.本项目的研究内容自获得资助以来，已在学术期刊上发表12篇论文，其中7篇SCI收录论文，5篇核心期刊，并均已标注本项目的编号。