【作者】 王芳；

【导师】 金光辉；

【作者基本信息】 厦门大学 ， 病理学与病理生理学， 2019， 硕士

【摘要】 放射性肺损伤是胸部放疗最常见并发症,包括早期放射性肺炎和晚期放射性肺纤维化。研究表明,放射性肺损伤的形成过程需经历上皮细胞间充质转化(EMT)。如今,肺癌患者的生存率正在增加,放射性肺损伤也随之引起广泛关注。MEN1基因编码核蛋白menin,menin与MLL等组蛋白修饰酶相互作用调节关键靶基因,参与调控多种细胞表型等。有研究表明,在乳腺癌细胞中,menin与RPA2相互作用,促进细胞增殖、黏附、转移、侵袭及EMT发生。提示menin在EMT进程中具有潜在的生物学功能。实验室前期研究表明,MEN1基因缺失抑制Kras突变诱导的EMT进程,提示menin参与调控肺癌EMT进程。然而,menin在放疗所致EMT进程中的作用机制目前尚不清楚。因此,本研究试图探讨menin在辐射诱导的肺EMT中的作用,并进一步探究menin是否通过EMT介导放射性肺损伤进程。基于以上科学问题,本课题构建了放射性肺损伤动物模型。分别得到野生型组、Men1单基因敲除组、单独辐照组及Men1基因敲除联合辐照组,且分别在辐射后8周及辐射后16周收取小鼠肺组织。通过免疫组化实验,发现辐射后8周出现放射性肺炎现象,说明在辐射8周时处于放射性肺损伤的炎症期。通过马松染色实验,发现辐射16周后出现放射性肺纤维化现象,说明在辐射16周时处于放射性肺损伤的纤维化期。通过HE染色及免疫组化实验,发现Men1单基因敲除后EMT进程加快,小鼠肺部炎症反应增加,说明Men1缺失加速放射性肺炎进程。通过马松染色及免疫组化实验,发现Men1单基因敲除后EMT进程加快,小鼠肺部纤维化反应增加,说明Men1缺失加速放射性纤维化进程。由于menin-MLL相互作用参与多种生物学功能,因此我们进一步探讨敲低menin加速放射性肺损伤是否与MLL作用有关。通过MLL基因敲除小鼠肺组织免疫组化检测,发现MLL单基因敲除后EMT水平被明显抑制,说明menin和MLL都参与辐射诱导的EMT进程,但作用机制不一定相同。综上所述,本研究发现Men1在放射性肺损伤进程中是关键的抑制基因,为预防和治疗放射性肺损伤提供了新的思路。更多还原

【Abstract】 Radiation-induced lung injury,the most common complication of chest radiotherapy.Studies have shown that the formation of radiation-induced lung injury undergoes epithelial mesenchymal transition(EMT).Nowadays the survival rate of lung cancer patients is increasing,and radiation-induced lung injury has also gathered attention a lot.The nuclear protein menin which is encoded by gene MEN],and interaction with histone modification enzymes such as mixed lineage lymphoma gene(MLL)to regulate key target genes and participating in the regulation of various cell phenotypes.Studies have shown that in breast cancer cells,menin interacts with RPA2 to promote cell proliferation,adhesion,metastasis,invasion and occur of EMT.It gives a clue that menin has potential biological function in the EMT process.In the pre-laboratory study,Kras mutation induced EMT in the process of lung adenocarcinoma,and MEN1 gene deletion inhibited the EMT process induced by Kras mutation.This suggests that menin is involved in the regulation of lung cancer EMT.However,the mechanism of action of menin in the EMT process caused by radiotherapy is still unclear.Therefore,this study sought to explore the role of menin in radiation-induced lung EMT and to investigate whether menin mediates the progression of radiation-induced lung injury via EMT.Based on the above scientific problems,animal models of radiation-induced lung injury were divided into wild type group,the Menl single gene knockout group,the single irradiation group and the Men]gene knockout combine irradiation group.The lung tissues of the mice were collected 8 weeks and 16 weeks after the irradiation respectively.Via immunohistochemistry experiments,It indicated that radioactive pneumonia occurred 8 weeks after irradiation,which showed that it was the inflammatory phase of radiation-induced lung injury during 8 weeks of irradiation.Masson staining experiment showed radioactive pulmonary fibrosis occurred after 16 weeks of radiation,indicating that the status was in the fibrosis stage of radiation-induced lung injury at 16 weeks of irradiation.By way of HE staining and immunohistochemistry experiments,it was found that the EMT process accelerated after knockout of Men1 gene,and mice’s lung inflammation increased.The 2 results indicated that Men1 deficiency accelerated the process of radiation pneumonitis.Through the Masson staining and immunohistochemistry experiments,it was found that the EMT process accelerated and the lung fibrosis of reaction increased in mice after knockout of the Men1 gene.These indicated that the Men1 deficiency accelerated the process of radioactive fibrosis.Since the menin-MLL interaction involved in various biological functions,it would be investigated that whether acceleration of radiation-induced lung injury caused by knockout of menin is related to MLL.It was found that EMT levels were significantly inhibited after MLL single gene knockout.It indicated that both menin and MLL are involved in the EMT process caused by radiation,but the mechanism of actions were not necessarily same.In summary,menin plays a key inhibitory role in the process of radiation-induced lung injury.This study provides new insights into the prevention and treatment of lung injury caused by radiotherapy.更多还原