电离辐射诱发miRNA表达谱改变及EGCG辐射防护分子机制的初步研究
发布时间：2019-03-06 11:59
【摘要】：电离辐射(ionizing radiation,IR)可通过直接或间接作用造成机体损伤,microRNA(miRNA)是一类非编码小RNA,与电离辐射损伤密切相关。表没食子茶素没食子酸酯(epigallocatechin gallate,EGCG)是茶多酚中抗氧化活性最高的成分,主要通过清除自由基等方式发挥辐射防护作用。目前关于EGCG的研究多集中在辐射防护方面,而进一步的辐射防护分子机制研究较少。为从miRNA角度探究EGCG的辐射防护分子机制,首先建立小鼠辐射损伤模型,采用高通量测序技术对小鼠体内的miRNA进行差异表达筛选。小鼠经一次性总剂量为4 Gy的60Co全身辐射后,其肝脏中共鉴定出48个差异表达的miRNA,包括20个表达上调的miRNA以及28个表达下调的miRNA。辐射后小鼠胸腺中差异表达的miRNA共有112个,其中77个miRNA上调,35个miRNA下调。qRT-PCR验证结果与测序结果一致。靶基因预测结果显示,小鼠体内差异表达miRNA的靶基因主要参与基因的复制、重组与修复、信号转导机制、转录调控机制等生命活动,并参与小鼠的相关肝代谢途径以及小鼠胸腺中影响T淋巴细胞成熟的信号通路。根据高通量测序结果,以miR-34a作为研究对象进一步探究EGCG的体内外辐射防护分子机制。在本文中,CCK8活力检测结果显示,经不同浓度EGCG(0、5、10、20、50、100μM)预处理24h后,AML-12细胞活力显著性上升,且在辐射2或4 Gy后培养0、12 h,相比于辐射组,其细胞活力显著性提高(p0.05),连续培养24 h后细胞活力呈剂量依赖性上升(p0.05)。qRT-PCR结果显示,与对照组相比,电离辐射引发AML-12细胞中miR-34a表达量显著性上调(0.001p0.01),经不同浓度的EGCG预处理后,miR-34a表达量与辐射组相比均显著性下调(p0.05)。AML-12细胞经转染miR-mimics后,miR-34a表达量显著提高且可明显抑制Sirt1表达(p0.05)。小鼠经4 Gy辐射后,与对照组相比,其肝脏中miR-34a表达量显著性上调(p0.01),Sirt1表达量显著性下调(p0.001)。灌喂不同浓度(30、60、120 mg/kg BW·d)的EGCG后,与辐射组相比,小鼠肝脏中miR-34a表达量显著性下调且呈剂量依懒性(p0.05),Sirt1表达上调(p0.05)。本论文主要利用高通量测序技术筛选电离辐射引发的差异表达的miRNA,并以miR-34a作为研究对象初步探究EGCG的辐射防护分子机制,结果说明电离辐射能够影响小鼠体内miRNA的差异表达,且小鼠胸腺中差异表达的miRNA更多;机体可通过miRNA调控靶基因参与多种复杂的生物学网络从而进行自我修复与防护;EGCG可促进AML-12细胞增殖,且有效提高其辐射后的细胞活力,并且可能通过抑制miR-34a促进Sirt1表达量从而减少辐射引发的细胞凋亡,保护机体免受电离辐射损伤。
[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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q691.5
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