电离辐射作用后溶酶体介导的细胞死亡机制研究

The irreparable damage caused by radiation could trigger cell death. The previous research on cell death mostly focused on the upstream signal pathways, while in this study we will center about lysosome-mediated cell death in pulmonary epithelial cells, epidermal cells and breast cells. Firstly, the radiation-induced lysosome membrane permeabilization (LMP), release of Cathepsins and cell death will be observed; Bafilomycin A1and chloroquine will be used to inhibit lysosomal activity and Siramesine used for LMP, to analyze if the cell death is lysosome-dependent; Secondly, whether lysosome-mediated autophagy affects the final death rate will be determined. The effect of radiation on autophagy and cell death will be compared with those under hypoxia and starvation condition as well as oxidative stress, the autophagy inhibitor 3-MA will then be used to determine the contribution of autophagy to cell death. Thirdly, the effects of radiation on lysosome turnover, autolysosome trafficking, lysosome activities will be detected. the roles of cathepsins in radiation-induced cell death will be also detected in the presence of cathepsins inhibitors or cathepsins-shRNA. Fourthly, the lysosome-dependent mitochondrial outer membrane permeabilization (MOMP), mitochondrial fragmentation, Bax clustering and the release of Cyt c will be detected after radiation. Fifthly, the association between lysosome status and cell death will be observed, the LMP inducer and inhibitor will be used in xenograft to determine if they function as radiosensitizer or not. This study will supplement and enrich the theory on radiation damage and radiation-induced cell death.

放射诱导细胞死亡机制的研究多集中在调控通路。本项目以溶酶体为核心开展下游机制研究，即溶酶体介导的细胞死亡。选择辐射损伤常见的肺上皮细胞、表皮细胞和易受放疗累及的乳腺细胞：（1）观察辐射损伤后溶酶体膜通透（LMP）、水解酶Cathepsin释放、细胞死亡，进而通过抑制溶酶体功能或诱导LMP，确定细胞死亡是否依赖于溶酶体。（2）确定LMP调节自噬后能否影响细胞死亡率，比较辐射效应与自噬模型（乏氧、饥饿）、氧化应激模型（H2O2）中自噬和细胞死亡情况；给予自噬抑制剂3-MA确定自噬对细胞死亡的影响。（3）观察辐射对溶酶体蛋白酶活性、周转和自噬溶酶体Trafficking的影响。（4）观察辐射对溶酶体依赖性线粒体膜通透（MOMP）、线粒体片段化、Bax集簇化等的影响。（5）观察肿瘤患者中溶酶体状态与细胞死亡关系，应用LMP调控剂于荷瘤鼠模型中观察辐射增敏作用。本研究将丰富辐射致细胞死亡的理论。

辐射诱导细胞损伤和死亡一直以来是放射生物学的研究热点，本项目首次以溶酶体为核心开展下游机制研究，即溶酶体介导的细胞死亡。我们研究发现：1）电离辐射引起溶酶体膜以剂量和时间依赖的方式通透性增加； 2)溶酶体介导的细胞死亡主要是依赖于铁和ROS的自噬性细胞死亡；3)溶酶体介导的细胞死亡不依赖于溶酶体释放的Cathepsin；4）电离辐射首先诱导的LMP进而促进线粒体损伤；5）线粒体损伤剂TTFA显著增加辐射诱导的细胞死亡，而抗氧化剂NAC抑制辐射诱导的线粒体损伤和细胞死亡；6）溶酶体铁参与了辐射诱导的细胞死亡的调控机制；7）沉默线粒体蛋白BNIP3促进辐射诱导的细胞死亡；8）电离辐射引起不同肿瘤细胞的死亡方式不同；9）溶酶体破坏剂可与化疗药物协同引起细胞死亡。.本研究将正常细胞与肿瘤细胞进行比较研究，从全新的溶酶体损伤机制入手，不同于以往研究辐射损伤仅针对于正常细胞进行，目的是了解两者机制的不同更有利于针对性对正常组织进行辐射防护，对肿瘤组织进行增敏杀伤。考虑到将可能的机制用于肿瘤杀伤，具有转化医学的特色，对指导临床应用有重要价值。