黄芪对重离子辐射诱发肺癌相关MSCs旁效应及基因组不稳定的防护机制研究

Heavy ion is known as the best ray for cancer radiotherapy in the 21st century.But its bystander effects get growing concern. Mesenchymal stem cells (MSCs) is one of the major interstitial cells in the tumor microenvironment. Our previous study has shown that Astragalus can enhance resistance of MSCs which are induced to arise malignant transformation in the tumor microenvironment. This study prepares to establish the tumor microenvironment of MSCs and lung cancer cell lines A549 in vivo and in vitro,which are irradiated by carbon ion beam. Using modern biological technology to detect①proliferation,differentiation,cytoskeleton,mult- iple differentiation, paracrine capabilities, micronucleus formation rate and DNA damage repair and so of MSCs and progeny cells in the A549 microenvironment; Using biochip to analyze expressions of differential genes;②Irradiated MSCs,A549 and tumor cause bystander effects on unirradiated MSCs in vivo and in vitro in proliferation,differentiation and changes of genomic instability,etc; ③Expres- sions of ROS,NOS,NO and changes of DNA methylation for analyzing induced bystander effect mechanism in MSCs;④Based on past and present clinical unilateral application experience of Astragalus, we observe the influence of the intervention groups on these indexes. In order to ascertain straightforward and bystander effects of heavy ion beams on cancer-related MSCs and protection mechanisms of Astragalus to provide the scientific basis for heavy ion therapy optimization.

重离子被誉为21世纪放疗治癌最理想射线，但其旁效应等非靶效应日益受到关注。骨髓间充质干细胞（MSCs）是肿瘤微环境的主要间质细胞之一，课题组前期已证明黄芪可增强MSCs对肿瘤微环境诱发恶性转化的抗性。本课题拟建立MSCs与肺癌细胞株A549体内外肿瘤微环境，经碳离子束辐照后，检测①A549微环境中MSCs及子代细胞增殖分化、细胞骨架、多向分化性、旁分泌能力及微核形成率、DNA损伤修复等基因组不稳定性的影响，利用生物芯片分析差异基因表达；②受辐射MSCs、A549、瘤体对未受辐射体内外MSCs增殖分化、基因组不稳定性变化等旁效应；③ MSCs中ROS、NOS、NO表达水平及DNA甲基化变化，分析诱发MSCs旁效应机制；④基于古今临床黄芪单方应用经验，分组干预观察黄芪对上述指标的影响。以期探明重离子束对癌相关MSCs的直接或旁效应及其黄芪防护机制，为重离子治癌优化方案提供科学依据。

为了探讨肺癌辐射相关间充质干细胞（MSCs）的旁效应，本研究从直接辐射、转移条件培养基、体内辐射肺部肿瘤动物模型三个体系中观察了肺癌相关MSCs的生物学变化规律，以细胞增殖与凋亡、细胞微核和53BP1 foci焦点形成水平等作为生物学终点，探讨与信号分子TGF-β、ROS和MAPK、NF-κB通路蛋白有关的发生机制。黄芪多糖（APS）干预后，研究APS对MSCs增殖、染色体损伤、DNA损伤的防护作用，信号分子TGF-β、ROS的变化水平以及对MAPK和NF-κB信号通路的调节作用，为肺癌放疗的安全性及辐射防护提供科学依据。结果显示MSCs增殖异常、微核和Foci点的形成，可能与TGF-β和ROS信号分子激活胞内MAPK/NF-κB通路蛋白有关；体内肺癌局部辐照时，旁器官中MSCs会受影响，可能与TGF-β和ROS信号分子活化有关。TGF-β和ROS信号分子活化胞内MAPK/NF-κB通路蛋白可能是旁细胞MSCs体内外损伤的机制。APS对MSCs的基因组不稳定性等旁效应损伤具有保护作用，可以通过调节TGF-β、ROS信号分子和MAPK、NF-κB通路蛋白来发挥保护作用。