本文主要研究内容
作者宋志全（2019）在《电离辐射对DNA损伤修复蛋白DNA-PKcs及其结合RNA作用的研究》一文中研究指出：目的:环境中电离辐射对人体细胞基因组DNA造成最严重的一种损害是DNA双链断裂(DNA double strand breaks,DSBs),当发生DSBs时,人体细胞最主要的修复通路是以DNA-PKcs蛋白(DNA-dependent Protein Kinase Catalytic Subunit)为核心因子主导的DNA非同源性末端连接(nonhomologous end joining,NHEJ)。近年来,对DNA-PKcs进行了大量研究,DNA-PKcs作为上游的关键蛋白激酶在NHEJ通路中发挥重要的作用。因此,我们关注电离辐射致人体细胞基因双链断裂损伤后DNA-PKcs蛋白激酶活性的变化,研究其激酶活性增强具有辐射时间剂量效应,从而可以作为人体细胞基因放射损伤修复机制研究的靶蛋白。随着近年来不断有研究表明DNA-PKcs蛋白可以通过结合RNA发挥功能,我们进一步从RNA-蛋白复合体角度研究DNA-PKcs蛋白在电离辐射损伤修复过程中的作用。方法:(1)在室温条件下使用本院60 Coγ射线照射构建细胞DSBs模型。8Gy剂量照射U2OS细胞,分别收取未照射对照组(con)和照后(0.5、1、2、4、8 h)时间点的细胞,通过Western Blots实验观察电离辐射后不同时间点DNA-PKcs蛋白及其Ser2056位点磷酸化表达情况;接下来对U2OS细胞进行不同剂量(0,2,4,8 Gy)60Coγ射线照射处理,收取2h时间点细胞,Western Blots实验观察DNA-PKcs蛋白及其Ser2056位点磷酸化表达情况。同时为了排除细胞特异性实验采用不同剂量(0,2,4,8 Gy)的60Coγ射线照射HeLa细胞、293T细胞、HepG2细胞,Western Blots实验观察照射后2h时间点各细胞中DNA-PKcs蛋白Ser2056位点磷酸化表达情况。(2)以DNA-PKcs为目标蛋白,实验用8Gy 60Coγ射线照射U2OS细胞,分别提取未照射对照组(con)和照射后(0.5、1、2、4、8 h)各个时间点样品核蛋白,对其分别进行特异性RNA结合蛋白免疫沉淀,分离提取沉淀中蛋白质和RNA,Western Blots实验验证抗体蛋白富集效果,利用RNA分光光度仪和电泳实验对免疫沉淀复合物中分离纯化的RNA质量进行初步检验;高通量测序分析(诺禾致源公司)实验备选RNA样品,对于测序数据结果,使用Fast QC进行质量控制与统计,通过BWA软件与参考基因进行比对,使用cufflink软件拼接比对结果并计算表达量。通过差异分析将对照组(con)分别和实验照射组(0.5h、1h)差异表达基因的交集作为研究与DNA-PKcs蛋白结合RNA的目标基因组。然后对目标基因组进行功能注释(GO注释和KEGG注释),筛选出富集度较高的RNA分子作为后续研究的候选RNA并进行qRT-PCR实验表达验证,选出差异大的RNA作为最后研究对象。使用siRNA抑制目标RNA表达的方法进行功能实验,同时选用敲低DNA-PKcs表达和使用DNA-PKcs激酶活性抑制剂的实验组进行结果对照。结果:(1)60Coγ射线照射后DNA-PKcs蛋白激酶活性增强具有时间效应。(2)60Coγ射线照射后DNA-PKcs蛋白激酶活性增强具有剂量效应。(3)60Coγ射线照射对DNA-PKcs蛋白激酶活性的剂量效应不具有细胞特异性。(4)实验所选DNA-PKcs抗体能够有效的免疫沉淀DNA-PKcs蛋白。(5)实验分离纯化获得的RNA分光光度计测得OD260/280范围在1.8-2.2,凝胶电泳质检RNA有较完整、清晰的RNA条带。(6)筛选出KEGG通路分析及GO富集分析共同选取的富集度高的3条代谢通路,3条富集通路交集有5个基因分别是ITGA3、ITGA5、ITGAV、CD44和SDC4。(7)目标基因在qRT-PCR实验表达均有增高趋势,其中ITGA5和SDC4基因表达差异最明显。(8)使用siRNA抑制ITGA5、SDC4基因表达,实验中细胞迁移率均明显降低,实验结果与通过敲低DNA-PKcs表达或使用DNA-PKcs激酶活性抑制剂导致细胞迁移率降低趋势具有一致性。结论:(1)研究了人体细胞DNA-PKcs蛋白激酶活性表达具有电离辐射的时间和剂量效应,因此DNA-PKcs蛋白可作为人体细胞基因放射损伤修复机制研究的靶蛋白。(2)成功获得了与U2OS细胞DNA-PKcs蛋白复合体结合的RNA高通量测序库。(3)DNA-PKcs蛋白与ITGA5、SDC4基因可能在细胞运动能力方面有相互调节作用。
Abstract
mu de :huan jing zhong dian li fu she dui ren ti xi bao ji yin zu DNAzao cheng zui yan chong de yi chong sun hai shi DNAshuang lian duan lie (DNA double strand breaks,DSBs),dang fa sheng DSBsshi ,ren ti xi bao zui zhu yao de xiu fu tong lu shi yi DNA-PKcsdan bai (DNA-dependent Protein Kinase Catalytic Subunit)wei he xin yin zi zhu dao de DNAfei tong yuan xing mo duan lian jie (nonhomologous end joining,NHEJ)。jin nian lai ,dui DNA-PKcsjin hang le da liang yan jiu ,DNA-PKcszuo wei shang you de guan jian dan bai ji mei zai NHEJtong lu zhong fa hui chong yao de zuo yong 。yin ci ,wo men guan zhu dian li fu she zhi ren ti xi bao ji yin shuang lian duan lie sun shang hou DNA-PKcsdan bai ji mei huo xing de bian hua ,yan jiu ji ji mei huo xing zeng jiang ju you fu she shi jian ji liang xiao ying ,cong er ke yi zuo wei ren ti xi bao ji yin fang she sun shang xiu fu ji zhi yan jiu de ba dan bai 。sui zhao jin nian lai bu duan you yan jiu biao ming DNA-PKcsdan bai ke yi tong guo jie ge RNAfa hui gong neng ,wo men jin yi bu cong RNA-dan bai fu ge ti jiao du yan jiu DNA-PKcsdan bai zai dian li fu she sun shang xiu fu guo cheng zhong de zuo yong 。fang fa :(1)zai shi wen tiao jian xia shi yong ben yuan 60 Coγshe xian zhao she gou jian xi bao DSBsmo xing 。8Gyji liang zhao she U2OSxi bao ,fen bie shou qu wei zhao she dui zhao zu (con)he zhao hou (0.5、1、2、4、8 h)shi jian dian de xi bao ,tong guo Western Blotsshi yan guan cha dian li fu she hou bu tong shi jian dian DNA-PKcsdan bai ji ji Ser2056wei dian lin suan hua biao da qing kuang ;jie xia lai dui U2OSxi bao jin hang bu tong ji liang (0,2,4,8 Gy)60Coγshe xian zhao she chu li ,shou qu 2hshi jian dian xi bao ,Western Blotsshi yan guan cha DNA-PKcsdan bai ji ji Ser2056wei dian lin suan hua biao da qing kuang 。tong shi wei le pai chu xi bao te yi xing shi yan cai yong bu tong ji liang (0,2,4,8 Gy)de 60Coγshe xian zhao she HeLaxi bao 、293Txi bao 、HepG2xi bao ,Western Blotsshi yan guan cha zhao she hou 2hshi jian dian ge xi bao zhong DNA-PKcsdan bai Ser2056wei dian lin suan hua biao da qing kuang 。(2)yi DNA-PKcswei mu biao dan bai ,shi yan yong 8Gy 60Coγshe xian zhao she U2OSxi bao ,fen bie di qu wei zhao she dui zhao zu (con)he zhao she hou (0.5、1、2、4、8 h)ge ge shi jian dian yang pin he dan bai ,dui ji fen bie jin hang te yi xing RNAjie ge dan bai mian yi chen dian ,fen li di qu chen dian zhong dan bai zhi he RNA,Western Blotsshi yan yan zheng kang ti dan bai fu ji xiao guo ,li yong RNAfen guang guang du yi he dian yong shi yan dui mian yi chen dian fu ge wu zhong fen li chun hua de RNAzhi liang jin hang chu bu jian yan ;gao tong liang ce xu fen xi (nuo he zhi yuan gong si )shi yan bei shua RNAyang pin ,dui yu ce xu shu ju jie guo ,shi yong Fast QCjin hang zhi liang kong zhi yu tong ji ,tong guo BWAruan jian yu can kao ji yin jin hang bi dui ,shi yong cufflinkruan jian pin jie bi dui jie guo bing ji suan biao da liang 。tong guo cha yi fen xi jiang dui zhao zu (con)fen bie he shi yan zhao she zu (0.5h、1h)cha yi biao da ji yin de jiao ji zuo wei yan jiu yu DNA-PKcsdan bai jie ge RNAde mu biao ji yin zu 。ran hou dui mu biao ji yin zu jin hang gong neng zhu shi (GOzhu shi he KEGGzhu shi ),shai shua chu fu ji du jiao gao de RNAfen zi zuo wei hou xu yan jiu de hou shua RNAbing jin hang qRT-PCRshi yan biao da yan zheng ,shua chu cha yi da de RNAzuo wei zui hou yan jiu dui xiang 。shi yong siRNAyi zhi mu biao RNAbiao da de fang fa jin hang gong neng shi yan ,tong shi shua yong qiao di DNA-PKcsbiao da he shi yong DNA-PKcsji mei huo xing yi zhi ji de shi yan zu jin hang jie guo dui zhao 。jie guo :(1)60Coγshe xian zhao she hou DNA-PKcsdan bai ji mei huo xing zeng jiang ju you shi jian xiao ying 。(2)60Coγshe xian zhao she hou DNA-PKcsdan bai ji mei huo xing zeng jiang ju you ji liang xiao ying 。(3)60Coγshe xian zhao she dui DNA-PKcsdan bai ji mei huo xing de ji liang xiao ying bu ju you xi bao te yi xing 。(4)shi yan suo shua DNA-PKcskang ti neng gou you xiao de mian yi chen dian DNA-PKcsdan bai 。(5)shi yan fen li chun hua huo de de RNAfen guang guang du ji ce de OD260/280fan wei zai 1.8-2.2,ning jiao dian yong zhi jian RNAyou jiao wan zheng 、qing xi de RNAtiao dai 。(6)shai shua chu KEGGtong lu fen xi ji GOfu ji fen xi gong tong shua qu de fu ji du gao de 3tiao dai xie tong lu ,3tiao fu ji tong lu jiao ji you 5ge ji yin fen bie shi ITGA3、ITGA5、ITGAV、CD44he SDC4。(7)mu biao ji yin zai qRT-PCRshi yan biao da jun you zeng gao qu shi ,ji zhong ITGA5he SDC4ji yin biao da cha yi zui ming xian 。(8)shi yong siRNAyi zhi ITGA5、SDC4ji yin biao da ,shi yan zhong xi bao qian yi lv jun ming xian jiang di ,shi yan jie guo yu tong guo qiao di DNA-PKcsbiao da huo shi yong DNA-PKcsji mei huo xing yi zhi ji dao zhi xi bao qian yi lv jiang di qu shi ju you yi zhi xing 。jie lun :(1)yan jiu le ren ti xi bao DNA-PKcsdan bai ji mei huo xing biao da ju you dian li fu she de shi jian he ji liang xiao ying ,yin ci DNA-PKcsdan bai ke zuo wei ren ti xi bao ji yin fang she sun shang xiu fu ji zhi yan jiu de ba dan bai 。(2)cheng gong huo de le yu U2OSxi bao DNA-PKcsdan bai fu ge ti jie ge de RNAgao tong liang ce xu ku 。(3)DNA-PKcsdan bai yu ITGA5、SDC4ji yin ke neng zai xi bao yun dong neng li fang mian you xiang hu diao jie zuo yong 。
论文参考文献
论文详细介绍
论文作者分别是来自军事科学院的宋志全，发表于刊物军事科学院2019-09-02论文，是一篇关于电离辐射论文,双链断裂论文,免疫共沉淀论文,高通量测序论文,军事科学院2019-09-02论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自军事科学院2019-09-02论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：电离辐射论文; 双链断裂论文; 免疫共沉淀论文; 高通量测序论文; 军事科学院2019-09-02论文;