橙皮苷和橙皮素对电离辐射致小鼠心血管损伤的防护作用研究

本研究以C57BL/6J小鼠为对象,探讨橙皮苷和橙皮素对其电离辐射致心血管损伤的防护作用。将小鼠随机分为空白对照组(NC组)、照射对照组(IR组)、橙皮苷低剂量组(L组200 mg/kg)、橙皮苷中剂量组(M组400 mg/kg)、橙皮苷高剂量组(H组800 mg/kg)和橙皮素低剂量组(L组50 mg/kg)、橙皮素中剂量组(M组100 mg/kg)、橙皮素高剂量组(H组200 mg/kg)8个组。照前2 h采用灌胃方式,空白对照组和照射对照组给予等量溶媒,除空白对照组外,各组小鼠均接受单次2 Gy的^(60)Coγ射线照射。分别于照射后24 h和48 h,对小鼠的免疫、抗氧化和DNA损伤三方面指标进行检测。结果显示,与照射组相比,给予橙皮苷和橙皮素的小鼠的肝脏指数均显著降低,但未表现出明显的剂量-效应关系,说明橙皮苷和橙皮素可能对辐射损伤小鼠的器官具有一定的保护作用;给予橙皮苷和橙皮素的小鼠血清中IL-1β、IL-6等炎性因子均显著降低,且具有剂量-效应关系,高剂量组表现更突出;给予橙皮苷和橙皮素均可显著降低小鼠主动脉丙二醛(MDA)含量和过氧化氢(H_(2)O_(2))含量,表明两种处理具有显著的抗氧化作用;给予橙皮苷和橙皮素能降低照射后小鼠主动脉组织γ-H2AX foci数量,且具有剂量依赖性关系,表明两种处理能减轻照射后小鼠主动脉DNA的损伤。本研究的结果表明橙皮苷和橙皮素对照射致小鼠血管损伤具有一定的保护作用。

人IER5基因在昆虫杆状病毒表达系统中的表达与鉴定

目的:利用昆虫杆状病毒表达系统表达早期快速反应基因5(IER5)蛋白,为后续蛋白质结晶及探索蛋白质三级结构提供线索。方法:以HeLa细胞cDNA为模板扩增出IER5基因片段,构建重组转移质粒pFastBac1-IER5;重组转移质粒经双酶切及测序鉴定后转化至感受态细胞DH10Bac,以获得重组的穿梭质粒rBacmid-IER5;将重组穿梭质粒感染Sf9细胞,待细胞出现明显病变时收集重组杆状病毒;用间接免疫荧光、SDS-PAGE及Western blot以及对表达产物进行分析鉴定。结果:重组转移质粒pFastBac1-IER5经双酶切后得到与预期相同的2条条带;重组穿梭质粒rBacmid-IER5经PCR鉴定在3300 bp左右出现一条特异性条带;间接免疫荧光结果提示IER5蛋白在Sf9细胞中得到表达;SDS-PAGE结果显示,表达产物的相对分子质量约为48 k,Western blot结果表明表达产物能与IER5抗体特异性结合。结论:利用昆虫-杆状病毒表达系统成功表达了人IER5蛋白,获得了体外表达重组IER5蛋白的相对分子质量、溶解性等物理化学特性。

宫颈癌细胞放射损伤后IER5蛋白表达及其相互作用蛋白的筛选和验证

的 了解IER5蛋白经放射损伤后的表达变化,并筛选IER5蛋白在放射损伤后可能参与DNA损伤修复通路中的相互作用蛋白.方法 HeLa细胞经4 Gyγ射线照射后在不同时间点收集细胞,提取总蛋白质和细胞核蛋白行Western blot检验;构建3×Flag标签融合表达的IER5蛋白表达载体,转染人肾上皮细胞293T并照射,收集细胞行免疫沉淀富集IER5的结合蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离免疫共沉淀复合物,考马斯亮蓝染色观察差异条带并切胶酶解后行质谱分析,对可能相互作用蛋白结果进行Western blot验证.结果 IER5蛋白照后4h表达逐渐增加,12 h至高峰,持续至48 h;细胞核内IER5蛋白表达也有增加趋势;质谱分析数据表明,照射组检测出374个蛋白,对照组检测出256个蛋白,两组比较差异蛋白41个,照射组中包括与DNA结合、代谢、损伤修复相关的10个蛋白;其中,与DNA损伤修复密切相关的1型聚ADP核糖基化聚合酶（PARP1）得到Western blot验证.结论 IER5是放射损伤相关蛋白,其可能参与DNA损伤修复通路.

DNA damage stress induces the dissociation of Smurf1/2 from MDM2 in a slow manner

The tumor suppressor p53 locates at the key point of cell growth or apoptosis balance, and the expression level of p53 is tightly controlled by ubiquitin ligases including MDM2. Upon DNA damage stresses, p53 was accumulated and activated, leading to cell cycle arrest or apoptosis. We previously showed that Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by interacting with and stabilizing MDM2, and consequently enhancing MDM2-mediated ubiquitylation of p53. However, it is unclear how the Smurf1-MDM2 interaction is regulated in response to DNA damage stress. Here, we show that in response to etoposide treatment Smurf1 dissociates from MDM2, resulting in MDM2 destabilization and p53 accumulation. The negative regulation of Smurf1 on apoptosis is released. Notably, this dissociation is a slow process rather than a rapid response, implicating high expression of Smurf1 might confer the resistance against p53 activation. Consistent with this notion, we observed that Smurf1/2 ligases are highly expressed in colon cancer, esophageal squamous cell carcinoma and pancreatic cancer tissues, suggesting the oncogenic tendency of Smurf1/2.

p53-dependent upregulation of PIG3 transcription by γ-ray irradiation and its interaction with KAP1 in responding to DNA damage

PIG3 (p53-inducible gene 3), originally identified as one of a set of genes induced by p53 before the onset of apoptosis, was assumed to contribute to early cellular response to DNA damage. Here, we studied the relation between p53 status and the increased expression of PIG3 by ionizing radiation (IR), and the related clues regarding the involvement of PIG3 in the cellular response to IR-induced DNA damage signaling. We demonstrated that the pentanucleotide microsatellite sequence was responsible for the p53-dependent induction of PIG3 transcription after irradiation, while sequence upstream of PIG3 promoter could maintain the basal level of expression which was not inducible by irradiation. The interaction of PIG3 and the KRAB-ZFP-associated protein 1 (KAP1), a DNA damage response protein, was revealed. PIG3 nucleus foci were formed 15 min after γ-ray irradiation, and which were found to partially colocalize with the phospho-KAP-1 foci as well as γ-H2AX foci. Although the lac operator tagged EGFP based reporter system revealed that PIG3 does not remodel chromatin in large scale in the cells under normal growing condition, it indeed prompted the chromatin relaxation in the cellular response to DNA damage signaling. All these data suggest that PIG3 is involved in IR-induced DNA damage response, and which maybe partially attribute to its interaction with KAP1.

Clustered DNA damage induced by protons radiation in plasmid DNA

Clustered DNA damage is considered as a critical type of lesions induced by ionizing radiation, which can be converted into the fatal or strong mutagenic complex double strand breaks (DSBs) during damage processing in the cells. The new data show that high energy protons produce more potentially lethal DSBs than low LET radiation. In this study, plasmid DNA were used to in-vestigate and re-evaluate the biological effects induced by the protons with the LET of ～3.6 keV/μm at the molecular level in vitro, including single strand breaks (SSBs), DSBs, isolated and clustered base damages. The results of complex DNA damage detections indicated that protons at the given LET value induce about 1.6 fold more non-DSB clustered DNA damages than the prompt DSB. The DNA damage yields by protons were greater than that by γ-rays, specifically by 6 fold for the isolated type of DNA damage and 14 fold for the clustered damage. Furthermore, the spectrum of damages was also demonstrated to be depended on the radiation quality, with protons producing more DSBs relative to clusters than do γ-rays.

放射性肺损伤小鼠晚期转录水平特征性基因标志物的研究

目的研究不同剂量X射线照射小鼠肺部后的差异基因并进行转录水平分析,探讨小鼠放射性肺损伤(RILI)潜在的基因标志物。方法8周龄C57BL6雌性小鼠96只,X射线单次照射分为3组(12只/组):对照组(未接受照射)、中等剂量组(MD组,10 Gy单次照射)、高剂量组(HD组,20 Gy单次照射),余60只分为5组进行X射线全肺野梯度剂量照射。采用全基因组表达芯片对小鼠肺组织进行RNA检测并生成基因表达矩阵,使用R语言软件包进行基因表达数据转换,对特征性基因标志物表达水平进行验证与数学建模,对HD组小鼠基因表达水平进行基因本体论生物学功能基因集富集分析。采用经典贝叶斯检验方法进行组间基因表达差异的比较,采用线性回归模型分析2组独立数据的关联程度(关联系数为R2)。结果MD组和HD组小鼠照射后肺组织转录组学分析,共筛选出RILI差异表达基因539个,其中差异最显著的5个基因为Phlda3、Fgg、Kng1(上调基因)和Ptprb、Kit(下调基因)。梯度剂量X射线分次照射实验结果证实,3个上调基因均随X线剂量的增加而表达增强;2个下调基因则随剂量的上升而表达降低。上调基因的逻辑回归模型拟合程度较高(χ^2=11.66, R2=0.88);下调基因的表达值与剂量具有良好的相关性(R=-0.95,R2=0.89)。基因集富集分析结果显示,上调基因富集于p53信号通路、固有免疫反应等生物学过程,下调基因富集于细胞代谢、肺部发育等生物学过程。结论上调基因Phlda3、Fgg、Kng1与下调基因Ptprb、Kit可作为客观反应RILI严重程度的潜在基因标志物,为日后开展临床与辐射防护相关应用奠定前临床基础。

电离辐射损伤相关长链非编码RNA研究进展

长链非编码RNA （lncRNAs）是一类新的功能分子，可通过影响基因转录、蛋白质翻译以及蛋白质稳定性等方式调节下游靶基因，在生长发育、免疫应答、代谢调控以及肿瘤形成等生物学事件中发挥重要作用。已有研究表明lncRNAs可以通过电离辐射诱导表达，并参与细胞对电离辐射的应答反应以及细胞损伤修复过程。通过对电离辐射相关lncRNAs的研究有助于加深对电离辐射损伤应答机制的认识和了解。笔者对lncRNAs的结构功能、调控靶基因方式以及对电离辐射相关lncRNAs的功能和作用方式进行综述。