乏氧条件下辐射诱导人支气管上皮细胞旁效应中DNA甲基化改变的研究

Direct ionized radiation for cancer therapy can cause cell damage on peripheral unirradiated normal tissue cells, this phenomenon is called radiation-induced bystander effect which can be triggered by low dosage and produce widely ranged impacts. Bystander effect can induce DNA methylation, whichincreasing genomic instability and mutation rate. Tissue damages caused by bystander effect are more serious under hypoxic condition, and hypoxiaoftenexists for tumor tissues. Lung cancer is the most popular malignant cancer in Yunnan. Therefore, human bronchial epithelium is the target cells used in this research to investigate the changes and mechanism of DNA methylation under hypoxic conditions of radiation-induced bystander effect. Whole genome methylation sequencing technique is employed to analyses the DNA methylation differences of radiation induced effective bronchial epithelium under hypoxic and aerobic conditions and discover the key genetic DNA methylation alternations induced by bystander effect under hypoxic condition; the different methylation regions are verified throughbisulfite sequencing;Q-PCR analyses the enzymatic expression which associated with DNA methylation of effective cells; the level expression of ROS, NO and HIF-1 are detectedto explore the possible role ofoxidative metabolism in the radiation-induced bystander effect under hypoxic condition, also the DMSO and iNOS inhibitor aminoguanidine inhibition test are employed; according to the sequencing results and the usual tumor biological effectthe biological impacts of relative genes are evaluated. This research is beneficial to the future development of radiation protections, and reduces side effects of therapy outside radiation range; also the determination of occurrence mechanism of radiation-induced bystander effect is meaningfully important.

电离辐射可直接照射治疗肿瘤,还可致周围未受照组织产生同样损伤,即辐射诱导旁效应,其触发剂量低,影响范围广。旁效应可引起DNA甲基化改变,造成基因组不稳定性增加,基因突变率增高。乏氧造成的旁效应损伤更为严重,且被实质肿瘤所特有,肺癌是云南高发恶性肿瘤,因此研究以支气管上皮为对象,探讨乏氧条件下辐射诱导旁效应中DNA甲基化的改变及机制。用全基因组甲基化测序分析乏氧/有氧条件辐射诱导支气管上皮旁效应细胞DNA甲基化差异,焦磷酸测序验证关键基因DNA甲基化改变；Q-PCR检测乏氧下DNA甲基化相关酶表达；ROS、NO及HIF-1表达测定,DMSO与iNOS抑制实验探讨氧化代谢在乏氧下诱导旁效应的作用；根据测序结果获得的相关靶基因信息结合肿瘤常见生物学效应，检测细胞生物学影响。研究有利于今后开展辐射防护、降低治疗照射野外副作用,对辐射诱导旁效应发生机制的阐明也具有重要意义。

电离辐射可直接照射治疗肿瘤，还可致周围未受照组织产生同样损伤，即辐射诱导旁效应。接受放射治疗的患者中，周围未照射组织仍常常受到影响。肺脏是放射性敏感器官，在肺癌、霍奇金淋巴瘤、保乳术或乳腺癌切除术后辅助临床放射治疗中，周围肺组织常完全非目的性暴露于电离辐射中。在电离辐射继发的恶性肿瘤中，肺癌是最常见的实体肿瘤之一。因此，对辐射诱导旁效应的发生机制开展深入研究，并针对机制研发保护性抑制剂或激活剂，将有利于开展有效的辐射防护、降低治疗照射野外或全身的放射治疗的副作用。. 本研究通过DNA甲基化芯片及Q-PCR技术检测乏氧条件下辐射诱导人正常支气管上皮细胞旁效应模型细胞中DNA甲基化和相关甲基化酶的变化差异，同时检测旁效应模型细胞的细胞生物学改变和氧化代谢变化，分析旁效应模型细胞DNA甲基化芯片和表达谱芯片中相关基因通路的改变，寻求乏氧条件下辐射诱导旁效应发生的可能机制。结果发现乏氧条件下辐射诱导的旁效应细胞有DNA甲基化改变，且在辐射剂量0.5Gy下变化最大；乏氧辐射诱导的旁效应细胞呈现整体DNA高甲基化增强趋势，DNA甲基转移酶上调；旁效应细胞的克隆形成率降低、DNA损伤修复阻滞；DNA甲基化酶抑制剂能阻止乏氧和辐射对旁效应细胞的损伤。DNA甲基化芯片及表达谱芯片分析表明旁效应细胞中细胞间通讯、细胞内信号转导通路及DNA损伤修复相关基因的甲基化和表达受到影响。乏氧条件下辐射细胞上清中IL-6表达升高，进而抑制旁细胞中WNT5A和β-catenin基因表达，DNA甲基化酶抑制剂能阻止乏氧和辐射对旁效应细胞的损伤，阻止乏氧和辐射对WNT5A和β-catenin基因表达的抑制。本实验在国内首次研究乏氧条件下辐射诱导人支气管上皮细胞旁效应中DNA甲基化改变，有助于今后开展辐射防护、降低治疗照射野外副作用。