電離輻射誘發(fā)miRNA表達譜改變及EGCG輻射防護分子機制的初步研究
發(fā)布時間：2019-03-06 11:59
【摘要】：電離輻射(ionizing radiation,IR)可通過直接或間接作用造成機體損傷,microRNA(miRNA)是一類非編碼小RNA,與電離輻射損傷密切相關。表沒食子茶素沒食子酸酯(epigallocatechin gallate,EGCG)是茶多酚中抗氧化活性最高的成分,主要通過清除自由基等方式發(fā)揮輻射防護作用。目前關于EGCG的研究多集中在輻射防護方面,而進一步的輻射防護分子機制研究較少。為從miRNA角度探究EGCG的輻射防護分子機制,首先建立小鼠輻射損傷模型,采用高通量測序技術對小鼠體內的miRNA進行差異表達篩選。小鼠經一次性總劑量為4 Gy的60Co全身輻射后,其肝臟中共鑒定出48個差異表達的miRNA,包括20個表達上調的miRNA以及28個表達下調的miRNA。輻射后小鼠胸腺中差異表達的miRNA共有112個,其中77個miRNA上調,35個miRNA下調。qRT-PCR驗證結果與測序結果一致。靶基因預測結果顯示,小鼠體內差異表達miRNA的靶基因主要參與基因的復制、重組與修復、信號轉導機制、轉錄調控機制等生命活動,并參與小鼠的相關肝代謝途徑以及小鼠胸腺中影響T淋巴細胞成熟的信號通路。根據高通量測序結果,以miR-34a作為研究對象進一步探究EGCG的體內外輻射防護分子機制。在本文中,CCK8活力檢測結果顯示,經不同濃度EGCG(0、5、10、20、50、100μM)預處理24h后,AML-12細胞活力顯著性上升,且在輻射2或4 Gy后培養(yǎng)0、12 h,相比于輻射組,其細胞活力顯著性提高(p0.05),連續(xù)培養(yǎng)24 h后細胞活力呈劑量依賴性上升(p0.05)。qRT-PCR結果顯示,與對照組相比,電離輻射引發(fā)AML-12細胞中miR-34a表達量顯著性上調(0.001p0.01),經不同濃度的EGCG預處理后,miR-34a表達量與輻射組相比均顯著性下調(p0.05)。AML-12細胞經轉染miR-mimics后,miR-34a表達量顯著提高且可明顯抑制Sirt1表達(p0.05)。小鼠經4 Gy輻射后,與對照組相比,其肝臟中miR-34a表達量顯著性上調(p0.01),Sirt1表達量顯著性下調(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表達量顯著性下調且呈劑量依懶性(p0.05),Sirt1表達上調(p0.05)。本論文主要利用高通量測序技術篩選電離輻射引發(fā)的差異表達的miRNA,并以miR-34a作為研究對象初步探究EGCG的輻射防護分子機制,結果說明電離輻射能夠影響小鼠體內miRNA的差異表達,且小鼠胸腺中差異表達的miRNA更多;機體可通過miRNA調控靶基因參與多種復雜的生物學網絡從而進行自我修復與防護;EGCG可促進AML-12細胞增殖,且有效提高其輻射后的細胞活力,并且可能通過抑制miR-34a促進Sirt1表達量從而減少輻射引發(fā)的細胞凋亡,保護機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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：鄭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q691.5
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