本文選題：低劑量輻射 + 高本底輻射地區(qū)　； 參考：《中國疾病預(yù)防控制中心》2017年碩士論文

【摘要】：背景隨著醫(yī)學(xué)放射性檢查的普及,飛行活動的增加等長期低劑量輻射暴露與人們?nèi)粘Ｉ铌P(guān)系日益密切,低劑量輻射的健康效應(yīng)研究逐漸成為關(guān)注熱點�？煽康纳飿�(biāo)志物對于明確低劑量輻射的生物學(xué)效應(yīng)及作用機制具有關(guān)鍵作用。目前廣泛應(yīng)用的輻射效應(yīng)生物標(biāo)志物是以染色體畸變?yōu)榇淼募?xì)胞遺傳學(xué)指標(biāo),但它并不能全面反應(yīng)生物學(xué)效應(yīng),尤其是極低的照射劑量并不能引起細(xì)胞遺傳學(xué)指標(biāo)的變化,因此尋找適用范圍更廣泛、指示更靈敏的低劑量輻射生物標(biāo)志物具有重要意義。microRNA作為一類廣泛存在于真核生物的轉(zhuǎn)錄后調(diào)節(jié)因子,其表達(dá)水平不僅受到電離輻射的影響,還廣泛調(diào)控電離輻射相關(guān)的各種基因的表達(dá),進(jìn)而參與DNA損傷與修復(fù),細(xì)胞周期進(jìn)程改變,細(xì)胞凋亡等一系列輻射所致早期生物學(xué)效應(yīng)的調(diào)控。目的大量動物實驗和細(xì)胞實驗均表明電離輻射可以誘導(dǎo)miR-16、miR-106b、miR-34a、miR-449a和let-7g的表達(dá)改變,但對于它們在低劑量照射條件下的表達(dá)變化情況及調(diào)控機制還未明確。因此本研究在前期研究的基礎(chǔ)上,選擇高本底地區(qū)居民及AHH-1人B淋巴細(xì)胞分別進(jìn)行體內(nèi)和體外實驗研究,分析低劑量條件下外周血血漿和體外培養(yǎng)的淋巴細(xì)胞中5個miRNAs的表達(dá)變化,同時分析淋巴細(xì)胞內(nèi)miRNAs細(xì)胞周期相關(guān)靶基因和細(xì)胞周期的變化情況,為明確低劑量輻射所致的早期生物學(xué)效應(yīng)以及尋找效應(yīng)生物標(biāo)志物進(jìn)行初步探索。方法采用單純隨機抽樣方法分別從高本底地區(qū)和對照地區(qū)各選取55例女性居民作為調(diào)查對象,調(diào)查年齡、BMI等指標(biāo)并計算個人累積劑量。采用實時熒光定量PCR(RT-PCR)方法定量檢測研究對象外周血血漿中miR-106b、miR-16、miR-34a、miR-449a和let-7g的相對表達(dá)水平;同時提取外周血淋巴細(xì)胞,采用流式細(xì)胞術(shù)檢測細(xì)胞周期變化情況。模擬低劑量照射條件,分別用低劑量0.05Gy、0.075Gy、O.1Gy、0.2Gy和高劑量2Gy 60Co γ射線照射體外培養(yǎng)的AHH-1人B淋巴細(xì)胞,并分別于照射后4h,8h,12h,24h和48h采用實時熒光定量PCR定量檢測5個miRNAs的相對表達(dá)水平;應(yīng)用流式細(xì)胞術(shù)分析淋巴細(xì)胞中miRNAs調(diào)控的細(xì)胞周期相關(guān)靶基因Wee1、p21、CDK1、CyclinB1、Cdc25A的表達(dá)變化情況,以及細(xì)胞周期的改變情況。結(jié)果1、高本底地區(qū)研究結(jié)果(1)與對照組居民相比,高本底地區(qū)居民外周血血漿中miR-16和miR-106b的表達(dá)水平降低,miR-449a的表達(dá)水平升高(Z=-2.968、-2.089、-2.043,P0.05),差異具有統(tǒng)計學(xué)意義�？刂颇挲g等混雜因素后,血漿中miR-16和miR-106b的相對表達(dá)水平與個人累積劑量呈負(fù)相關(guān)關(guān)系(β=0.243、0.208,P0.05)。尚不能認(rèn)為高本底暴露能引起miR-34a和1et-7g在血漿中的表達(dá)改變(Z=-0.414、-1.832,P0.05)。(2)與對照組相比,高本底組外周血淋巴細(xì)胞中G0/G1期細(xì)胞所占比例降低,S期細(xì)胞占比增加(t=3.408、-3.412,P0.05)。2、AHH-1人B淋巴細(xì)胞研究結(jié)果(1)照射后 12h 分析 AHH-1 人 B 淋巴細(xì)胞中 miR-16,miR-106b,miR-34a,miR-449a和let-7g在各劑量組間相對表達(dá)水平的總體差異均有統(tǒng)計學(xué)意義(χ2=22.128、36.341、31.207、40.498、24.298,P0.05),隨著照射劑量的增加,5個miRNAs的表達(dá)水平均呈現(xiàn)上升的趨勢。5個miRNAs具有相似的時間變化趨勢,在8h出現(xiàn)表達(dá)低谷,在12h或24h時達(dá)到峰值。與假照組相比,0.075Gy劑量照射后 12h 時 miR-16,miR-106b,miR-34a,miR-449a 和 let-7g 分別升高至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淋巴細(xì)胞在受到低劑量照射后12h和48h時G2/M期的細(xì)胞占比增加(F=489.559、17.152,P0.05),2Gy照射后出現(xiàn)明顯的G2/M期阻滯。分析時程效應(yīng)結(jié)果表明在48h時G2/M期阻滯程度明顯降低,由12h時的42.77%降低至 7.76%。(3)與假照組相比,照射后12h時AHH-1人B淋巴細(xì)胞中p21,CDK1,CyclinB1 和 Cdc25A 在照射后表達(dá)相對下降(F=7.957、7.934、7.833、4.040,P0.05)。24h時分析結(jié)果表明Wee1,CDK1,CyclinB1和Cdc25A在照射后表達(dá)相對下降(F=5.425、12.241、9.577、9.584,P0.05)。結(jié)論1、高本底地區(qū)居民外周血血漿中miR-16和miR-106b表達(dá)下調(diào),且與個人累積劑量呈負(fù)相關(guān)關(guān)系,未發(fā)現(xiàn)高本底暴露能引起血漿中miR-449a,miR-34a和let-7g的表達(dá)改變。低劑量照射能誘導(dǎo)AHH-1人B淋巴細(xì)胞中miR-16,miR-106b,miR-34a,miR-449a 和 let-7g 的表達(dá)上調(diào)。2、低劑量輻射能引起外周血淋巴細(xì)胞S期細(xì)胞比例和AHH-1淋巴細(xì)胞中G2/M期細(xì)胞比例增加,提示低劑量電離輻射能誘導(dǎo)細(xì)胞出現(xiàn)DNA損傷介導(dǎo)的細(xì)胞周期進(jìn)程的適度改變。3、低劑量照射可誘導(dǎo)AHH-1淋巴細(xì)胞中細(xì)胞周期相關(guān)靶基因Wee1,p21,CDK1,CyclinB1 和 Cdc25A 表達(dá)下調(diào),說明 miR-16,miR-106b,miR-34a,miR-449a和let-7g及其靶基因可能參與了低劑量電離輻射誘導(dǎo)的細(xì)胞周期進(jìn)程調(diào)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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R818

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