【作者】 李玉文；

【导师】 刘青杰；

【作者基本信息】 中国疾病预防控制中心 ， 放射医学， 2009， 硕士

【摘要】 背景与目的:研究发现辐射可以诱导mtDNA突变和mtDNA拷贝数的变化,而突变的mtDNA和mtDNA拷贝数的改变都可进一步导致线粒体基因表达的改变,进而影响细胞的正常功能。然而目前有关辐射诱导线粒体基因表达的研究较少,且研究结果十分不一致。为此,本研究以人淋巴细胞株为研究对象,探索⁶⁰Coγ射线照射后线粒体基因表达改变的量效关系和时效关系,同时观察照射后呼吸链酶复合体Ⅳ（COX）活性的改变,以期摸索出辐射敏感的线粒体基因及其表达的时效、量效关系,为后期探索放射生物学领域中快速剂量估算这一难题提供基础理论依据。方法:1.以剂量率为1Gy/min的⁶⁰Coγ射线进行人淋巴细胞株的照射,分析量效关系时,剂量分别为1、3、5、8和10Gy,照射后培养8h;分析时效关系时剂量为5Gy,照射后分别培养0.5、4、8、12、24、48和72h。2.探索转录水平上辐射对淋巴细胞线粒体基因表达的影响时,首先利用逆转录PCR方法分别摸索13种线粒体基因的最佳PCR实验条件,然后利用Real-time PCR方法定量检测线粒体基因表达水平。3.探索翻译水平上辐射对淋巴细胞线粒体基因表达的影响时,利用流式细胞仪检测线粒体COXⅠ、COXⅡ和COXⅢ蛋白表达水平。4.线粒体COX活性的检测是通过分光光度计测定550nm处COX吸收峰值的改变来反映COX活性的变化。结果:1.⁶⁰Coγ射线照射人淋巴细胞后,线粒体基因在转录水平上的表达改变:量效关系方面,1～10Gy剂量照射后淋巴细胞线粒体基因表达普遍上调,大部分基因在3或8Gy剂量照射后表达增强较明显,COXⅠ、COXⅢ和ND3基因在3Gy剂量范围内呈现明显的量效关系。COXⅡ、ND2、ND5、Cytb和ATPase8基因在8Gy剂量范围内呈现明显的量效关系;时效关系方面,5Gy剂量照射后,除COXⅠ基因表达下调外,其余基因表达总体上调,照射后4h左右表达增强最显著,24h左右表达上调也十分明显,且大部分基因可持续高表达至48或72h。2.⁶⁰Coγ射线照射人淋巴细胞后,线粒体COX的三个亚基在翻译水平上的表达改变:当辐射剂量达到3.5Gy时,COXⅠ和Ⅱ蛋白表达明显增强;当5Gy剂量照射后4-8h,COXⅠ和Ⅱ蛋白表达才明显上调,而COXⅢ蛋白表达普遍下调。3.⁶⁰Coγ射线照射剂量达到3Gy时,才出现COX活性显著减弱,5Gy剂量照射0.5h后COX活性持续减弱。结论:1.辐射可以诱导人淋巴细胞线粒体基因表达的改变。2.在转录水平上,辐射可以诱导人淋巴细胞13种线粒体基因表达总体上调,其中COXⅢ和ND3基因在3Gy剂量范围内呈现十分良好的量效关系。照射后4h左右线粒体基因表达增强最显著,24h左右表达上调也十分明显,且大部分基因可持续高表达至48或72h。3.在翻译水平上,辐射可以诱导人淋巴细胞线粒体COXⅠ和COXⅡ蛋白表达总体上调,COXⅢ蛋白表达显著下调。4.辐射可以诱导人淋巴细胞线粒体COX活性持续降低。更多还原

【Abstract】 Background and Objective:A number of studies indicated that irradiation can induce mtDNA mutation and changes in mtDNA copy number,which can contribute to the changes of mitochondrial genes expression.However,the relationship between irradiation and mitochondrial genes expression has rarely been studied,and the results of investigations are varied.For these reasons,we explore the alterations in expression level of human lymphoblastoid cell mitochondrial genes and the activities of respiratory chain complexesⅣinduced by ⁶⁰Coγ-rays.Methods:1.Human lymphoblastoid cell lines were irradiated with ⁶⁰Coγ-rays at dose rate of 1Gy/min.The cells were cultured 8h after irradiation of 1～10Gy absorbed dose in order to detect the dose-effect relationship between the dose and expression changes of 13 mitochondrial genes,which encoded peptides.While the expression changes of these genes were analyzed at different post-irradiation time-points between 0.5 and 72h after irradiation of 5Gy in order to explore the time-effect relationship.2.Changes in mitochondrial genes mRNA level were detected by Real-time PCR after the PCR parameters were qualified by RT-PCR.3.Changes in mitochondrial genes COXⅠ,COXⅡand COXⅢprotein expression level were determined by flow cytometry（FCM）.4.Spectrophotometric analysis was applied to detect the COX activities.Results:1.The expression level of 13 mitonchondrial genes mRNAs in human lymphocytes were generally up-regulated after 1～10Gy irradiation,and the expression peak of most genes were at 3 or 8Gy exposure.The expression changes of COXⅠ, COXⅢand ND3 genes showed the dose-effect relationship after 1-3 Gy ⁶⁰Coγ-rays exposure;COXⅡ,ND2,ND5,Cyt b and ATPase8 gene expression changes showed the dose-effect relationship after 1-8 Gy exposure.Meanwhile the expression levels of mitochondrial genes mRNAs were elevated most significantly by 4h after 5Gy radiation except COXⅠ.2.Expression changes of COXⅠand COXⅡprotein were increased significantly after 3-5Gy exposure,while alterations in COXⅠand COXⅡprotein expression were elevated significantly 4-8h after 5Gy irradiation.In addition,COXⅢprotein expression levels were down-regulated generally.3.Activity of COX was found to be decreased significantly after exposure to 3-10Gy.It decreased significantly at 4-72h after exposure to 5Gy irradiation.Conclusions:1.Ionizing radiation might induce significant changes in mitochondrial gene expression.2.The expression levels of human lymphoblastoid cell mitochondrial genes mRNAs are generally up-regulated following ⁶⁰Coγ-rays irradiation and the expression changes of COXⅢand ND3 genes show the excellent dose-effect relationship after 1-3 Gy ⁶⁰Coγ-rays exposure.The expression peak changes of the 13 mitochondrial genes are at 4 h after irradiation.3.The expression levels of COXⅠand COXⅡprotein are increased comprehensively,while COXⅢprotein expression level is decreased generally after ⁶⁰Coγ-rays exposure.4.Activity of COX was decreased generally after ⁶⁰Coγ-rays exposure.更多还原