T-bet(+)GATA-3(+)Th1/2杂合细胞在放射性肺损伤中的作用及机制研究

Radiation-induced lung injury (RILI) is a major complication of thoracic radiotherapy. It be regarded as a continuous progression of events, which contains two discernable phase named radiation pneumonia and radiation pulmonary fibrosis. At present, the mechanisms involved in RILI remain incompletely understood. In the previous research, we found that the immune imbalance of Th1/Th2 exist in the progress of RILI. Further study showed that a type of cell called Th1/2 hybrid cells which co-expresses T-bet and GATA-3 that express obviously in both radiation pneumonia period(Th1 reaction period) and radiation pulmonary fibrosis period(Th2 reaction period). Since the Th1/2 hybrid cells can simultaneously secrete both IFN-γ and IL-4 to regulate the immune balance, we propose that there is a regulatory mechanism in the RILI that on the quantitative modulation of Th1/2 hybrid cells to the prevention of excessive Th1 or Th2 response. In this study, we first will use C57BL/6 mice to establish models of tumor-bearing and tumor-free RILI. By using flow cytometry analysis, we will to confirm the dynamic relationship between Th1,Th2 and T-bet(+)GATA-3(+)Th1/2 hybrid cells in these mice’s lung and blood before and after radiation. Then we will use immunofluorescence, smFISH and flow cytometry analysis to explore the regulatory mechanism of T-bet(+)GATA-3(+)Th1/2 hybrid cells in radiation-induced lung injury. T-bet(+)GATA-3(+)Th1/2 hybrid cells will be culturing in vitro and injecting into mice to clarify the regulating effect of T-bet(+)GATA-3(+)Th1/2 hybrid cells on immune imbalance of Th1/Th2 in RILI. This study aims at exploring the new immunoregulation mechanism of radiation-induced lung injury and probably immunotherapy method.

放射性肺损伤是肺癌放疗的常见并发症，目前该病的损伤机制仍未明确。既往我们发现放射性肺损伤中存在Th1/Th2免疫失衡，进一步研究发现在放射性肺损伤的两期，即早期放射性肺炎期（Th1型反应）和晚期肺纤维化期（Th2型反应）均存在一种T-bet(+)GATA-3(+)Th1/2杂合细胞。为探讨该杂合细胞在放射性肺损伤中的免疫调节功能，本项目拟通过构建荷瘤及无瘤小鼠放射性肺损伤模型，采用流式细胞术探讨放射性肺损伤中Th1、Th2和T-bet(+)GATA-3(+)Th1/2杂合细胞间的动态变化关系；通过免疫荧光, smFISH, 流式细胞学检测明确T-bet(+)GATA-3(+)Th1/2杂合细胞在放射性肺损伤中的调节机制；通过体外诱导T-bet(+)GATA-3(+)Th1/2杂合细胞干预探讨该杂合细胞对放射性肺损伤的调控作用。旨在探索放射性肺损伤中的免疫调控机制和新的免疫治疗途径。

放射性肺损伤(RILI)是胸部肿瘤放疗的常见并发症，严重影响了患者的疗效和生存质量，临床上表现为早期放射性肺炎和晚期不可逆性放射性肺纤维化。课题组前期研究发现放射性肺损伤初期产生以Th1为主的优势应答，放射性肺炎后期及纤维化期产生以Th2为主的优势应答。进一步研究发现放射性肺损伤过程中存在Th1/Th2免疫失衡。本课题组建立了荷瘤(Lewis肺癌)小鼠放射性肺损伤模型，观察了不同时间点该模型与其正常小鼠放疗对照组肺组织中Th1/Th2转录因子T-bet、GATA-3基因及蛋白水平的差异以及血清相关细胞因子表达情况。荷瘤放肺与无瘤放肺小鼠模型的脾组织CD4+T细胞比例存在明显差异，提示了荷瘤状态对小鼠的免疫状态有很严重的影响，且放疗对正常和荷瘤小鼠的免疫系统的刺激作用程度存在差异。Lewis肺癌小鼠放射性肺损伤模型中放射性肺纤维化的发生时间皆早于正常小鼠放射性肺损伤模型且更严重。其后通过检测照射后不同时间点肺组织中Th1、Th2的特异性转录因子T-bet、GATA-3的表达量变化证实 Lewis肺癌小鼠放射性肺损伤模型中同样存在Th1/Th2免疫失衡，且向更偏向于Th2反应，更趋向于肺纤维化，提示了肿瘤体质下确实更容易出现肺纤维化，且程度更重，这一现象与机体Th2免疫偏移有关，可作为临床放射性肺损伤肺纤维化不可逆的部分解释。另一方面通过适当提高Th1免疫可以减轻瘤小鼠及正常小鼠放射性肺损伤的发生，缓解放射性肺纤维化恶化。同时，我们通过野生型与Nrf2基因敲除鼠模型的肺部损伤及的肺部炎性细胞浸润、血清细胞因子、氧化损伤指标、抗氧化能力水平对比发现Nrf2过表达增强抗氧化能力减轻氧化损伤，对小鼠放射性肺损伤模型有保护作用，为该疾病的防治提供新的理论依据。