電離輻射誘發(fā)miRNA表達(dá)譜改變及EGCG輻射防護(hù)分子機(jī)制的初步研究
發(fā)布時(shí)間：2019-03-06 11:59
【摘要】：電離輻射(ionizing radiation,IR)可通過(guò)直接或間接作用造成機(jī)體損傷,microRNA(miRNA)是一類非編碼小RNA,與電離輻射損傷密切相關(guān)。表沒(méi)食子茶素沒(méi)食子酸酯(epigallocatechin gallate,EGCG)是茶多酚中抗氧化活性最高的成分,主要通過(guò)清除自由基等方式發(fā)揮輻射防護(hù)作用。目前關(guān)于EGCG的研究多集中在輻射防護(hù)方面,而進(jìn)一步的輻射防護(hù)分子機(jī)制研究較少。為從miRNA角度探究EGCG的輻射防護(hù)分子機(jī)制,首先建立小鼠輻射損傷模型,采用高通量測(cè)序技術(shù)對(duì)小鼠體內(nèi)的miRNA進(jìn)行差異表達(dá)篩選。小鼠經(jīng)一次性總劑量為4 Gy的60Co全身輻射后,其肝臟中共鑒定出48個(gè)差異表達(dá)的miRNA,包括20個(gè)表達(dá)上調(diào)的miRNA以及28個(gè)表達(dá)下調(diào)的miRNA。輻射后小鼠胸腺中差異表達(dá)的miRNA共有112個(gè),其中77個(gè)miRNA上調(diào),35個(gè)miRNA下調(diào)。qRT-PCR驗(yàn)證結(jié)果與測(cè)序結(jié)果一致。靶基因預(yù)測(cè)結(jié)果顯示,小鼠體內(nèi)差異表達(dá)miRNA的靶基因主要參與基因的復(fù)制、重組與修復(fù)、信號(hào)轉(zhuǎn)導(dǎo)機(jī)制、轉(zhuǎn)錄調(diào)控機(jī)制等生命活動(dòng),并參與小鼠的相關(guān)肝代謝途徑以及小鼠胸腺中影響T淋巴細(xì)胞成熟的信號(hào)通路。根據(jù)高通量測(cè)序結(jié)果,以miR-34a作為研究對(duì)象進(jìn)一步探究EGCG的體內(nèi)外輻射防護(hù)分子機(jī)制。在本文中,CCK8活力檢測(cè)結(jié)果顯示,經(jīng)不同濃度EGCG(0、5、10、20、50、100μM)預(yù)處理24h后,AML-12細(xì)胞活力顯著性上升,且在輻射2或4 Gy后培養(yǎng)0、12 h,相比于輻射組,其細(xì)胞活力顯著性提高(p0.05),連續(xù)培養(yǎng)24 h后細(xì)胞活力呈劑量依賴性上升(p0.05)。qRT-PCR結(jié)果顯示,與對(duì)照組相比,電離輻射引發(fā)AML-12細(xì)胞中miR-34a表達(dá)量顯著性上調(diào)(0.001p0.01),經(jīng)不同濃度的EGCG預(yù)處理后,miR-34a表達(dá)量與輻射組相比均顯著性下調(diào)(p0.05)。AML-12細(xì)胞經(jīng)轉(zhuǎn)染miR-mimics后,miR-34a表達(dá)量顯著提高且可明顯抑制Sirt1表達(dá)(p0.05)。小鼠經(jīng)4 Gy輻射后,與對(duì)照組相比,其肝臟中miR-34a表達(dá)量顯著性上調(diào)(p0.01),Sirt1表達(dá)量顯著性下調(diào)(p0.001)。灌喂不同濃度(30、60、120 mg/kg BW·d)的EGCG后,與輻射組相比,小鼠肝臟中miR-34a表達(dá)量顯著性下調(diào)且呈劑量依懶性(p0.05),Sirt1表達(dá)上調(diào)(p0.05)。本論文主要利用高通量測(cè)序技術(shù)篩選電離輻射引發(fā)的差異表達(dá)的miRNA,并以miR-34a作為研究對(duì)象初步探究EGCG的輻射防護(hù)分子機(jī)制,結(jié)果說(shuō)明電離輻射能夠影響小鼠體內(nèi)miRNA的差異表達(dá),且小鼠胸腺中差異表達(dá)的miRNA更多;機(jī)體可通過(guò)miRNA調(diào)控靶基因參與多種復(fù)雜的生物學(xué)網(wǎng)絡(luò)從而進(jìn)行自我修復(fù)與防護(hù);EGCG可促進(jìn)AML-12細(xì)胞增殖,且有效提高其輻射后的細(xì)胞活力,并且可能通過(guò)抑制miR-34a促進(jìn)Sirt1表達(dá)量從而減少輻射引發(fā)的細(xì)胞凋亡,保護(hù)機(jī)體免受電離輻射損傷。
[Abstract]:Ionizing radiation (ionizing radiation,IR) can cause damage to organism by direct or indirect action., microRNA (miRNA) is a class of non-coding small RNAs, which is closely related to ionizing radiation damage. Epigallocatechin gallate (epigallocatechin gallate,EGCG) is the most active antioxidant in tea polyphenols, which plays a role in radiation protection by scavenging free radicals. At present, most of the studies on EGCG are focused on radiation protection, but further studies on the molecular mechanism of radiation protection are less. In order to explore the molecular mechanism of radiation protection of EGCG from the point of view of miRNA, a mouse model of radiation injury was established, and the differential expression of miRNA in mice was screened by high throughput sequencing technique. After a total dose of 4 Gy 60Co, 48 differentially expressed miRNA, were identified in the liver, including 20 up-regulated miRNA and 28 down-regulated miRNA.. There were 112 differentially expressed miRNA in mouse thymus after irradiation, of which 77 miRNA were up-regulated and 35 miRNA were down-regulated. The results of qRT-PCR were consistent with those of sequencing. The results of target gene prediction showed that the target gene that differentially expressed miRNA in mice was mainly involved in gene replication, recombination and repair, signal transduction mechanism, transcription regulation mechanism and other life activities. It also participates in the liver metabolism pathway of mice and the signal pathway of T lymphocyte maturation in mouse thymus. According to the results of high-throughput sequencing, the molecular mechanism of radiation protection of EGCG in vitro and in vivo was further investigated with miR-34a as the research object. In this study, the results of CCK8 activity test showed that after 24 hours of pretreatment with different concentrations of EGCG (0,5,10,20,50100 渭 M), the viability of AML-12 cells increased significantly, and cultured for 0, 12 hours after 2 or 4 Gy irradiation, compared with the radiation group. After 24 hours of continuous culture, the cell viability increased in a dose-dependent manner (p0.05). The results of qRT-PCR showed that compared with the control group, the cell viability increased in a dose-dependent manner (p0.05). The expression of miR-34a in AML-12 cells was significantly up-regulated by ionizing radiation (0.001p0.01), and was pretreated with EGCG at different concentrations. The expression of miR-34a in AML-12 cells was significantly lower than that in radiation group (p0.05), and the expression of miR-34a in AML-12 cells was significantly higher than that in radiation group (p0.05), and the expression of miR-34a was significantly inhibited (p0.05). Compared with the control group, the expression of miR-34a in liver increased significantly (p0.01) and the expression of Sirt1 decreased significantly (p0.001) in the liver of mice irradiated with 4 Gy. After EGCG of different concentrations (30,60120 mg/kg BW 路d), compared with the radiation group, the expression of miR-34a in the liver of mice decreased significantly (p0.05), and the expression of Sirt1 was up-regulated (p0.05). In this thesis, high-throughput sequencing technique was used to screen the differentially expressed miRNA, induced by ionizing radiation and to explore the molecular mechanism of radiation protection of EGCG with miR-34a as the research object. The results showed that ionizing radiation could affect the differential expression of miRNA in mice, and the differential expression of miRNA in mouse thymus was more than that in mouse thymus. Target genes can be regulated by miRNA to participate in a variety of complex biological networks for self-repair and protection. EGCG can promote the proliferation of AML-12 cells and effectively increase the viability of AML-12 cells after irradiation, and it may reduce the apoptosis induced by radiation by inhibiting the expression of miR-34a to promote the expression of Sirt1 and protect the body from ionizing radiation damage.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q691.5
[Abstract]:Ionizing radiation (ionizing radiation,IR) can cause damage to organism by direct or indirect action., microRNA (miRNA) is a class of non-coding small RNAs, which is closely related to ionizing radiation damage. Epigallocatechin gallate (epigallocatechin gallate,EGCG) is the most active antioxidant in tea polyphenols, which plays a role in radiation protection by scavenging free radicals. At present, most of the studies on EGCG are focused on radiation protection, but further studies on the molecular mechanism of radiation protection are less. In order to explore the molecular mechanism of radiation protection of EGCG from the point of view of miRNA, a mouse model of radiation injury was established, and the differential expression of miRNA in mice was screened by high throughput sequencing technique. After a total dose of 4 Gy 60Co, 48 differentially expressed miRNA, were identified in the liver, including 20 up-regulated miRNA and 28 down-regulated miRNA.. There were 112 differentially expressed miRNA in mouse thymus after irradiation, of which 77 miRNA were up-regulated and 35 miRNA were down-regulated. The results of qRT-PCR were consistent with those of sequencing. The results of target gene prediction showed that the target gene that differentially expressed miRNA in mice was mainly involved in gene replication, recombination and repair, signal transduction mechanism, transcription regulation mechanism and other life activities. It also participates in the liver metabolism pathway of mice and the signal pathway of T lymphocyte maturation in mouse thymus. According to the results of high-throughput sequencing, the molecular mechanism of radiation protection of EGCG in vitro and in vivo was further investigated with miR-34a as the research object. In this study, the results of CCK8 activity test showed that after 24 hours of pretreatment with different concentrations of EGCG (0,5,10,20,50100 渭 M), the viability of AML-12 cells increased significantly, and cultured for 0, 12 hours after 2 or 4 Gy irradiation, compared with the radiation group. After 24 hours of continuous culture, the cell viability increased in a dose-dependent manner (p0.05). The results of qRT-PCR showed that compared with the control group, the cell viability increased in a dose-dependent manner (p0.05). The expression of miR-34a in AML-12 cells was significantly up-regulated by ionizing radiation (0.001p0.01), and was pretreated with EGCG at different concentrations. The expression of miR-34a in AML-12 cells was significantly lower than that in radiation group (p0.05), and the expression of miR-34a in AML-12 cells was significantly higher than that in radiation group (p0.05), and the expression of miR-34a was significantly inhibited (p0.05). Compared with the control group, the expression of miR-34a in liver increased significantly (p0.01) and the expression of Sirt1 decreased significantly (p0.001) in the liver of mice irradiated with 4 Gy. After EGCG of different concentrations (30,60120 mg/kg BW 路d), compared with the radiation group, the expression of miR-34a in the liver of mice decreased significantly (p0.05), and the expression of Sirt1 was up-regulated (p0.05). In this thesis, high-throughput sequencing technique was used to screen the differentially expressed miRNA, induced by ionizing radiation and to explore the molecular mechanism of radiation protection of EGCG with miR-34a as the research object. The results showed that ionizing radiation could affect the differential expression of miRNA in mice, and the differential expression of miRNA in mouse thymus was more than that in mouse thymus. Target genes can be regulated by miRNA to participate in a variety of complex biological networks for self-repair and protection. EGCG can promote the proliferation of AML-12 cells and effectively increase the viability of AML-12 cells after irradiation, and it may reduce the apoptosis induced by radiation by inhibiting the expression of miR-34a to promote the expression of Sirt1 and protect the body from ionizing radiation damage.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q691.5
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