本文主要研究内容
作者林皓（2019）在《microRNA-7敲除小鼠模型的建立及雄性生殖力分析》一文中研究指出：microRNA-7是一种在各物种间序列高度保守的microRNA。已有研究表明,microRNA-7在疾病的发生、器官的发育、癌症的产生等生物活动中发挥着重要的作用,并且在内分泌器官中有较高水平的表达。然而,其在动物生殖发育过程中的作用及机理尚不明确。本研究利用CRISPR/Cas9基因编辑技术构建了 microRNA-7三种亚型的基因敲除小鼠模型,并对其雄性生殖力进行了分析,为进一步研究microRNA-7在动物生殖发育过程中的作用奠定了基础。首先,利用实时荧光定量PCR(Real-time PCR)检测了成年小鼠各组织器官中microRNA-7的表达水平。结果表明microRNA-7在垂体、下丘脑、肾上腺等内分泌组织中具有高水平的表达,并在睾丸、子宫、附睾等生殖器官中具有较高水平的表达。此外,原位杂交检测结果表明,microRNA-7在小鼠睾丸中的支持细胞及各阶段生殖细胞中均有表达。其次,利用 CRISPR/Cas9 基因编辑技术,构建了 microRNA-7a1、microRNA-7a2以及microRNA-7b基因敲除小鼠模型。通过采用目标基因PCR扩增、测序、Real-time PCR的检测方法,对小鼠模型进行鉴定,结果表明microRNA-7三种亚型基因敲除小鼠均为基因序列大片段缺失个体,且缺失位于microRNA-7成熟序列区域。microRNA-7三种亚型基因敲除小鼠的microRNA-7基因表达水平都有极显著的降低。利用T7EI法对基因敲除小鼠潜在脱靶基因进行检测,结果表明三种microRNA-7基因敲除小鼠模型中均不存在脱靶效应。对microRNA-7三种亚型基因敲除小鼠模型的表型分析,发现基因敲除小鼠的出生率和成活率较野生型小鼠相比无明显差异。microRNA-7a2基因敲除成年小鼠的睾丸、附睾、子宫组织的体积与重量较野生型相比有显著性的减小和降低。但microRNA-7a1和microRNA-7b基因敲除成年小鼠各组织器官重量和形态与野生型小鼠相比无明显差异。配种实验结果表明,microRNA-7a2基因的缺失会导致雄性和雌性小鼠的不育,然而microRNA-7a1和microRNA-7b基因敲除小鼠与野生型小鼠相比繁殖力无显著性差异。对microRNA-7a2基因敲除成年小鼠睾丸和附睾组织进行切片染色观察,结果显示,睾丸中部分曲细精管的生精上皮中有空洞产生,附睾组织结构无明显变化,但附睾尾管腔中精子的数量明显减少。对microRNA-7a2基因敲除雄性小鼠精子活力检测发现,其精子数目、精子运动能力都较野生型小鼠相比有显著性的降低。血清中促性腺激素检测表明,雄性microRNA-7a2基因敲除小鼠血清中促卵泡激素(FSH)与黄体生成素(LH)的激素水平均有显著性的下降。最后,为进一步研究microRNA-7a2在雄性小鼠生殖系统中的作用和功能,我们利用CRISPR/Cas9基因编辑技术成功的构建了 Loxp序列标记的microRNA-7a2的转基因小鼠模型(microRNA-7a2flox/flox),为microRNA-7a2条件性基因敲除小鼠模型的建立奠定了基础。本研究结果表明,利用CRISPR/Cas9基因编辑技术构建的microRNA-7三种亚型基因敲除小鼠模型具有高可靠性,可以用于microRNA-7功能及其作用机理的研究。另外,本研究结果表明microRNA-7a2的缺失会造成雄性小鼠不育,生殖系统功能的改变,说明microRNA-7a2在小鼠的生殖发育中发挥着重要的作用。
Abstract
microRNA-7shi yi chong zai ge wu chong jian xu lie gao du bao shou de microRNA。yi you yan jiu biao ming ,microRNA-7zai ji bing de fa sheng 、qi guan de fa yo 、ai zheng de chan sheng deng sheng wu huo dong zhong fa hui zhao chong yao de zuo yong ,bing ju zai nei fen bi qi guan zhong you jiao gao shui ping de biao da 。ran er ,ji zai dong wu sheng shi fa yo guo cheng zhong de zuo yong ji ji li shang bu ming que 。ben yan jiu li yong CRISPR/Cas9ji yin bian ji ji shu gou jian le microRNA-7san chong ya xing de ji yin qiao chu xiao shu mo xing ,bing dui ji xiong xing sheng shi li jin hang le fen xi ,wei jin yi bu yan jiu microRNA-7zai dong wu sheng shi fa yo guo cheng zhong de zuo yong dian ding le ji chu 。shou xian ,li yong shi shi ying guang ding liang PCR(Real-time PCR)jian ce le cheng nian xiao shu ge zu zhi qi guan zhong microRNA-7de biao da shui ping 。jie guo biao ming microRNA-7zai chui ti 、xia qiu nao 、shen shang xian deng nei fen bi zu zhi zhong ju you gao shui ping de biao da ,bing zai gao wan 、zi gong 、fu gao deng sheng shi qi guan zhong ju you jiao gao shui ping de biao da 。ci wai ,yuan wei za jiao jian ce jie guo biao ming ,microRNA-7zai xiao shu gao wan zhong de zhi chi xi bao ji ge jie duan sheng shi xi bao zhong jun you biao da 。ji ci ,li yong CRISPR/Cas9 ji yin bian ji ji shu ,gou jian le microRNA-7a1、microRNA-7a2yi ji microRNA-7bji yin qiao chu xiao shu mo xing 。tong guo cai yong mu biao ji yin PCRkuo zeng 、ce xu 、Real-time PCRde jian ce fang fa ,dui xiao shu mo xing jin hang jian ding ,jie guo biao ming microRNA-7san chong ya xing ji yin qiao chu xiao shu jun wei ji yin xu lie da pian duan que shi ge ti ,ju que shi wei yu microRNA-7cheng shou xu lie ou yu 。microRNA-7san chong ya xing ji yin qiao chu xiao shu de microRNA-7ji yin biao da shui ping dou you ji xian zhe de jiang di 。li yong T7EIfa dui ji yin qiao chu xiao shu qian zai tuo ba ji yin jin hang jian ce ,jie guo biao ming san chong microRNA-7ji yin qiao chu xiao shu mo xing zhong jun bu cun zai tuo ba xiao ying 。dui microRNA-7san chong ya xing ji yin qiao chu xiao shu mo xing de biao xing fen xi ,fa xian ji yin qiao chu xiao shu de chu sheng lv he cheng huo lv jiao ye sheng xing xiao shu xiang bi mo ming xian cha yi 。microRNA-7a2ji yin qiao chu cheng nian xiao shu de gao wan 、fu gao 、zi gong zu zhi de ti ji yu chong liang jiao ye sheng xing xiang bi you xian zhe xing de jian xiao he jiang di 。dan microRNA-7a1he microRNA-7bji yin qiao chu cheng nian xiao shu ge zu zhi qi guan chong liang he xing tai yu ye sheng xing xiao shu xiang bi mo ming xian cha yi 。pei chong shi yan jie guo biao ming ,microRNA-7a2ji yin de que shi hui dao zhi xiong xing he ci xing xiao shu de bu yo ,ran er microRNA-7a1he microRNA-7bji yin qiao chu xiao shu yu ye sheng xing xiao shu xiang bi fan shi li mo xian zhe xing cha yi 。dui microRNA-7a2ji yin qiao chu cheng nian xiao shu gao wan he fu gao zu zhi jin hang qie pian ran se guan cha ,jie guo xian shi ,gao wan zhong bu fen qu xi jing guan de sheng jing shang pi zhong you kong dong chan sheng ,fu gao zu zhi jie gou mo ming xian bian hua ,dan fu gao wei guan qiang zhong jing zi de shu liang ming xian jian shao 。dui microRNA-7a2ji yin qiao chu xiong xing xiao shu jing zi huo li jian ce fa xian ,ji jing zi shu mu 、jing zi yun dong neng li dou jiao ye sheng xing xiao shu xiang bi you xian zhe xing de jiang di 。xie qing zhong cu xing xian ji su jian ce biao ming ,xiong xing microRNA-7a2ji yin qiao chu xiao shu xie qing zhong cu luan pao ji su (FSH)yu huang ti sheng cheng su (LH)de ji su shui ping jun you xian zhe xing de xia jiang 。zui hou ,wei jin yi bu yan jiu microRNA-7a2zai xiong xing xiao shu sheng shi ji tong zhong de zuo yong he gong neng ,wo men li yong CRISPR/Cas9ji yin bian ji ji shu cheng gong de gou jian le Loxpxu lie biao ji de microRNA-7a2de zhuai ji yin xiao shu mo xing (microRNA-7a2flox/flox),wei microRNA-7a2tiao jian xing ji yin qiao chu xiao shu mo xing de jian li dian ding le ji chu 。ben yan jiu jie guo biao ming ,li yong CRISPR/Cas9ji yin bian ji ji shu gou jian de microRNA-7san chong ya xing ji yin qiao chu xiao shu mo xing ju you gao ke kao xing ,ke yi yong yu microRNA-7gong neng ji ji zuo yong ji li de yan jiu 。ling wai ,ben yan jiu jie guo biao ming microRNA-7a2de que shi hui zao cheng xiong xing xiao shu bu yo ,sheng shi ji tong gong neng de gai bian ,shui ming microRNA-7a2zai xiao shu de sheng shi fa yo zhong fa hui zhao chong yao de zuo yong 。
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论文详细介绍
论文作者分别是来自中国农业大学的林皓，发表于刊物中国农业大学2019-01-17论文，是一篇关于小鼠敲除模型论文,雄性生殖力论文,中国农业大学2019-01-17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，资料来自中国农业大学2019-01-17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。