低剂量辐射对miRNAs及细胞周期相关靶基因的影响

发布时间：2018-06-25 00:40

本文选题：低剂量辐射 + 高本底辐射地区　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：背景随着医学放射性检查的普及,飞行活动的增加等长期低剂量辐射暴露与人们日常生活关系日益密切,低剂量辐射的健康效应研究逐渐成为关注热点。可靠的生物标志物对于明确低剂量辐射的生物学效应及作用机制具有关键作用。目前广泛应用的辐射效应生物标志物是以染色体畸变为代表的细胞遗传学指标,但它并不能全面反应生物学效应,尤其是极低的照射剂量并不能引起细胞遗传学指标的变化,因此寻找适用范围更广泛、指示更灵敏的低剂量辐射生物标志物具有重要意义。microRNA作为一类广泛存在于真核生物的转录后调节因子,其表达水平不仅受到电离辐射的影响,还广泛调控电离辐射相关的各种基因的表达,进而参与DNA损伤与修复,细胞周期进程改变,细胞凋亡等一系列辐射所致早期生物学效应的调控。目的大量动物实验和细胞实验均表明电离辐射可以诱导miR-16、miR-106b、miR-34a、miR-449a和let-7g的表达改变,但对于它们在低剂量照射条件下的表达变化情况及调控机制还未明确。因此本研究在前期研究的基础上,选择高本底地区居民及AHH-1人B淋巴细胞分别进行体内和体外实验研究,分析低剂量条件下外周血血浆和体外培养的淋巴细胞中5个miRNAs的表达变化,同时分析淋巴细胞内miRNAs细胞周期相关靶基因和细胞周期的变化情况,为明确低剂量辐射所致的早期生物学效应以及寻找效应生物标志物进行初步探索。方法采用单纯随机抽样方法分别从高本底地区和对照地区各选取55例女性居民作为调查对象,调查年龄、BMI等指标并计算个人累积剂量。采用实时荧光定量PCR(RT-PCR)方法定量检测研究对象外周血血浆中miR-106b、miR-16、miR-34a、miR-449a和let-7g的相对表达水平;同时提取外周血淋巴细胞,采用流式细胞术检测细胞周期变化情况。模拟低剂量照射条件,分别用低剂量0.05Gy、0.075Gy、O.1Gy、0.2Gy和高剂量2Gy 60Co γ射线照射体外培养的AHH-1人B淋巴细胞,并分别于照射后4h,8h,12h,24h和48h采用实时荧光定量PCR定量检测5个miRNAs的相对表达水平;应用流式细胞术分析淋巴细胞中miRNAs调控的细胞周期相关靶基因Wee1、p21、CDK1、CyclinB1、Cdc25A的表达变化情况,以及细胞周期的改变情况。结果1、高本底地区研究结果(1)与对照组居民相比,高本底地区居民外周血血浆中miR-16和miR-106b的表达水平降低,miR-449a的表达水平升高(Z=-2.968、-2.089、-2.043,P0.05),差异具有统计学意义。控制年龄等混杂因素后,血浆中miR-16和miR-106b的相对表达水平与个人累积剂量呈负相关关系(β=0.243、0.208,P0.05)。尚不能认为高本底暴露能引起miR-34a和1et-7g在血浆中的表达改变(Z=-0.414、-1.832,P0.05)。(2)与对照组相比,高本底组外周血淋巴细胞中G0/G1期细胞所占比例降低,S期细胞占比增加(t=3.408、-3.412,P0.05)。2、AHH-1人B淋巴细胞研究结果(1)照射后 12h 分析 AHH-1 人 B 淋巴细胞中 miR-16,miR-106b,miR-34a,miR-449a和let-7g在各剂量组间相对表达水平的总体差异均有统计学意义(χ2=22.128、36.341、31.207、40.498、24.298,P0.05),随着照射剂量的增加,5个miRNAs的表达水平均呈现上升的趋势。5个miRNAs具有相似的时间变化趋势,在8h出现表达低谷,在12h或24h时达到峰值。与假照组相比,0.075Gy剂量照射后 12h 时 miR-16,miR-106b,miR-34a,miR-449a 和 let-7g 分别升高至2.548 倍,6.292 倍,6.408 倍,2.501 倍和 2.301 倍(t=-2.667、-3.366、-5.059、-3.827、-3.739,P0.05)。(2)AHH-1人B淋巴细胞在受到低剂量照射后12h和48h时G2/M期的细胞占比增加(F=489.559、17.152,P0.05),2Gy照射后出现明显的G2/M期阻滞。分析时程效应结果表明在48h时G2/M期阻滞程度明显降低,由12h时的42.77%降低至 7.76%。(3)与假照组相比,照射后12h时AHH-1人B淋巴细胞中p21,CDK1,CyclinB1 和 Cdc25A 在照射后表达相对下降(F=7.957、7.934、7.833、4.040,P0.05)。24h时分析结果表明Wee1,CDK1,CyclinB1和Cdc25A在照射后表达相对下降(F=5.425、12.241、9.577、9.584,P0.05)。结论1、高本底地区居民外周血血浆中miR-16和miR-106b表达下调,且与个人累积剂量呈负相关关系,未发现高本底暴露能引起血浆中miR-449a,miR-34a和let-7g的表达改变。低剂量照射能诱导AHH-1人B淋巴细胞中miR-16,miR-106b,miR-34a,miR-449a 和 let-7g 的表达上调。2、低剂量辐射能引起外周血淋巴细胞S期细胞比例和AHH-1淋巴细胞中G2/M期细胞比例增加,提示低剂量电离辐射能诱导细胞出现DNA损伤介导的细胞周期进程的适度改变。3、低剂量照射可诱导AHH-1淋巴细胞中细胞周期相关靶基因Wee1,p21,CDK1,CyclinB1 和 Cdc25A 表达下调,说明 miR-16,miR-106b,miR-34a,miR-449a和let-7g及其靶基因可能参与了低剂量电离辐射诱导的细胞周期进程调控。
[Abstract]:Background with the popularization of medical radioactivity, long term low dose radiation exposure has become increasingly closely related to people's daily life. The health effect of low dose radiation has become a hot topic. Reliable biomarkers play a key role in identifying the biological effects and mechanism of low dose radiation. At present, the widely used biomarkers of radiation effect are cytogenetic indicators represented by chromosome aberration, but it does not respond to biological effects in an all-round way, especially the very low dose of radiation does not cause changes in cytogenetic indicators. Therefore, it is necessary to find a more widely applicable and sensitive low dose radiation biomarker. .microRNA, as a kind of posttranscriptional regulation factor widely existed in eukaryotes, is not only affected by ionizing radiation, but also widely regulates the expression of various genes related to ionizing radiation, and then participates in a series of radiation caused by DNA damage and repair, cell cycle process change, cell apoptosis and so on. Objective the regulation of biological effects. Objective a large number of animal experiments and cell experiments showed that ionizing radiation could induce changes in the expression of miR-16, miR-106b, miR-34a, miR-449a and let-7g, but the changes in their expression and regulation mechanism under low dose irradiation were not clear. In vivo and in vitro, the human and AHH-1 B lymphocytes in the high base area were studied in vitro and in vitro. The changes in the expression of 5 miRNAs in the peripheral blood plasma and in vitro cultured lymphocytes under low dose were analyzed, and the changes of the target gene and the cell cycle of the miRNAs cell cycle in the lymphocytes were analyzed in order to clear the low dose radiation. The early biological effects and the finding effect biomarkers were preliminarily explored. Methods 55 female residents were selected from the high base area and the control area by simple random sampling, and the age, BMI and other indexes were investigated. The real time fluorescence quantitative PCR (RT-PCR) Fang Fading was used. The relative expression levels of miR-106b, miR-16, miR-34a, miR-449a and let-7g in peripheral blood plasma were measured, and peripheral blood lymphocytes were extracted and the cell cycle changes were detected by flow cytometry. Low dose irradiation conditions were simulated with low dose 0.05Gy, 0.075Gy, O.1Gy, 0.2Gy, and high dose 2Gy 60Co gamma ray irradiation body, respectively. AHH-1 human B lymphocytes were cultured in vitro, and the relative expression levels of 5 miRNAs were measured by real-time quantitative PCR in 4h, 8h, 12h, 24h and 48h, and the expression of cell cycle related target genes regulated by miRNAs in lymphocytes was analyzed by flow cytometry. Results 1, the results of the high background study (1) compared with the control group, the expression level of miR-16 and miR-106b in the peripheral blood plasma of the residents in the high base area decreased, the expression level of miR-449a increased (Z=-2.968, -2.089, -2.043, P0.05), and the difference had the significance of integration. After controlling the age and other confounding factors, the plasma miR-16 and miR-1 were found. The relative expression level of 06B was negatively correlated with the cumulative dose of individual (beta =0.243,0.208, P0.05). It is still not considered that high background exposure can cause the expression changes of miR-34a and 1et-7g in plasma (Z=-0.414, -1.832, P0.05). (2) compared with the control group, the proportion of G0/G1 phase cells in the peripheral blood lymphocytes of the high background group is lower, and the S cell ratio is compared. Increase (t=3.408, -3.412, P0.05).2, AHH-1 human B lymphocyte study results (1) 12h analysis of AHH-1 human B lymphocyte after 12h analysis of miR-16, miR-106b, miR-34a, and the overall difference in the level of relative expression between each dose group. In addition, the expression level of the 5 miRNAs showed an upward trend,.5 miRNAs had a similar time change trend, the expression of low vale in 8h, and the peak value at 12h or 24h. Compared with the false group, 0.075Gy dose irradiated 12h miR-16, miR-106b, miR-34a, miR-449a and 12h, respectively, to 2.548 times, 6.292 times, 6.408 times, 2.501, respectively. Double and 2.301 times (t=-2.667, -3.366, -5.059, -3.827, -3.739, P0.05). (2) the percentage of AHH-1 human B lymphocytes increased in G2/M phase when exposed to low doses of 12h and 48h. 42.77% to 7.76%. (3), compared with the sham group, the expression of p21, CDK1, CyclinB1 and Cdc25A in the B lymphocyte of AHH-1 people after irradiation was relatively decreased after irradiation (F=7.957,7.934,7.833,4.040, P0.05).24h after irradiation (F=7.957,7.934,7.833,4.040, P0.05).24h. 1, the expression of miR-16 and miR-106b in the peripheral blood plasma was downregulated and negatively correlated with the individual cumulative dose. The expression of miR-449a, miR-34a and let-7g in plasma was not found to be induced by high background exposure. Low dose irradiation could induce miR-16, miR-106b, miR-34a, miR-449a, and let-7g in AHH-1 human B lymphocytes. The proportion of S phase cells in peripheral blood lymphocytes and the proportion of G2/M phase cells in AHH-1 lymphocytes increased by low dose radiation, suggesting that low dose ionizing radiation can induce a moderate change in cell cycle process mediated by DNA damage and.3, and low dose irradiation can induce cell cycle related target gene Wee1 in AHH-1 lymphocytes. The expressions of p21, CDK1, CyclinB1 and Cdc25A are down regulated, indicating that miR-16, miR-106b, miR-34a, miR-449a and let-7g and their target genes may be involved in the regulation of cell cycle processes induced by low dose ionizing radiation.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R818

【参考文献】