本文主要研究内容
作者魏于栋（2019）在《DMRT1调控山羊雄性生殖干细胞分化、自我更新的机理》一文中研究指出：雄性生殖干细胞(male germ stem cell,mGSCs),同样被称作精原干细胞(spermatogonial stem cells,SSCs)是位于睾丸曲细精管基底膜上,既能通过自我更新维持自身群体数量的稳定,又能在体内定向分化并最终形成精子的一类成体干细胞。雄性生殖干细胞可以作为研究精子发生、减数分裂调控和细胞重编程的一个理想体外模型。Double sex and mab-3 related transcription factor 1(DMRT1)基因编码的蛋白质包含一个double sex/mab-3结构域,无论是在脊椎动物或者无脊椎动物中,这个最保守的结构域都会参与个体的性别决定过程。除此之外,DMRT1参与雄性生殖干细胞的损伤修复、抑制睾丸肿瘤、维持雄性生殖干细胞有丝分裂与减数分裂的平衡并且调控雄性生殖干细胞的多能性。因此,探究DMRT1在雄性生殖干细胞中的功能机制,可以为提高动物遗传育种、动物疾病模型和物种保护提供理论指导。在本研究中我们首次研究了DMRT1对山羊雄性生殖干细胞分化和自我更新过程中的影响,以及DMRT1在睾丸生殖免疫中发挥的功能。实验结果如下:1.DMRT1基因在哺乳动物中广泛存在且不同物种中序列高度保守,多序列比对的结果发现6种哺乳动物的DMRT1基因序列一致性达到了86.42%。对于其核心结构域DM结构域分析表明,不同物种的氨基酸序列基本一致,说明DM结构域在不同物种中同样具有高度保守性。为了探究DMRT1在山羊不同组织的表达情况,免疫荧光染色结果表明DMRT1在山羊睾丸中特异表达。染色及定量PCR进一步发现DMRT1在1月龄山羊睾丸的表达量很低,3月龄睾丸起始减数分裂后DMRT1表达明显上调,至个体成熟其表达水平趋于稳定。其次,利用免疫荧光共染色揭示了DMRT1主要定位在未分化和分化中的雄性生殖干细胞中,但是在启动减数分裂的精母细胞中却没有表达。根据以上结果,我们认为DMRT1主要在雄性生殖干细胞中发挥功能,对于mGSCs的分化和增殖发挥重要作用。2.FUW-TetO-Dmrt1慢病毒载体配合rtTA辅助质粒转染雄性生殖干细胞及永生化的mGSCs-I-SB细胞系,筛选出最适诱导DMRT1表达的Dox浓度。EDU、双荧光素酶检测、免疫荧光染色、Western blotting、及定量PCR进一步揭示DMRT1对于山羊雄性生殖干细胞发育过程的功能。在1μg/mL Dox诱导条件下,雄性生殖干细胞增殖能力及分化水平显著增高,减数分裂相关标记相应下调。维甲酸引起雄性生殖干细胞的减数分裂,但过表达DMRT1抑制减数分裂的发生同时mGSCs的增殖标记PCNA及分化标记c-KIT表达上调。因此DMRT1一方面促进雄性生殖干细胞的增殖和分化,另一方面抑制由RA引起的减数分裂异常起始,从而维持精原细胞有丝分裂和减数分裂的平衡。3.DMRT1在睾丸生殖免疫中发挥重要功能,DMRT1通过抑制TLRs信号通路从而降低雄性生殖干细胞中炎症反应的发生。shRNA干扰睾丸雄性生殖干细胞中DMRT1的表达,转录组测序及免疫组化试验结果显示mGSCs中TLR4及NF-κB被激活。ELISA及Western blotting检测发现雄性生殖干细胞内促炎因子TNFα及IL-6的表达上调。免疫共沉淀及双荧光素酶检测试验进一步揭示DMRT1招募PLZF蛋白,二者共同抑制TLR4和NF-κB的转录并促进雄性生殖干细胞数量的回补。与此同时,TUNEL染色及流式细胞术发现DMRT1抑制由炎症反应引起的雄性生殖干细胞的凋亡。综合以上结果表明DMRT1在促进雄性生殖干细胞自我更新及维持睾丸内环境稳态中的重要功能,提供了治疗睾丸生殖缺陷疾病的新思路。4.白消安处理的小鼠睾丸表现出严重的生殖细胞损伤,过表达DMRT1显著恢复了雄性生殖干细胞的数量并且维持精子发生的正常进行。免疫荧光染色及ELISA检测的结果表明DMRT1促进雄性生殖干细胞增殖标记PCNA、VASA及自我更新相关标记PLZF及CSF1R的表达。同时mGSCs自我更新相关的细胞因子GDNF、CSF1表达上调,从而证明DMRT1对睾丸内环境的重建至关重要。基于双分子荧光互补技术(BiFC)和免疫共沉淀检测,最终确认DMRT1蛋白与PLZF蛋白互作主要是由DMRT1与PLZF的BTB结构域互作来实现的。所以DMRT1与PLZF发挥协同作用参与雄性生殖干细胞自我更新下游通路的调节。除此之外,DMRT1和PLZF作为抑炎因子有效地抑制曲细精管内炎症的发生,为雄性生殖细胞损伤的修复建立了良好的微环境。综上所述,我们首次探究了DMRT1在雄性生殖干细胞中的表达模式,探索了DMRT1对山羊雄性生殖干细胞分化和自我更新过程的影响,并揭示DMRT1在生殖免疫中发挥的功能。基因修饰载体转染山羊雄性生殖干细胞,体内体外综合分析、验证筛选DMRT1可能作用的特异靶基因及其互作机制,为研究山羊雄性生殖干细胞的自我更新、精子发生的机理提供新的科学依据;为高产优质家畜的遗传、繁育和不育症的防治提供新方案。
Abstract
xiong xing sheng shi gan xi bao (male germ stem cell,mGSCs),tong yang bei chen zuo jing yuan gan xi bao (spermatogonial stem cells,SSCs)shi wei yu gao wan qu xi jing guan ji de mo shang ,ji neng tong guo zi wo geng xin wei chi zi shen qun ti shu liang de wen ding ,you neng zai ti nei ding xiang fen hua bing zui zhong xing cheng jing zi de yi lei cheng ti gan xi bao 。xiong xing sheng shi gan xi bao ke yi zuo wei yan jiu jing zi fa sheng 、jian shu fen lie diao kong he xi bao chong bian cheng de yi ge li xiang ti wai mo xing 。Double sex and mab-3 related transcription factor 1(DMRT1)ji yin bian ma de dan bai zhi bao han yi ge double sex/mab-3jie gou yu ,mo lun shi zai ji chui dong wu huo zhe mo ji chui dong wu zhong ,zhe ge zui bao shou de jie gou yu dou hui can yu ge ti de xing bie jue ding guo cheng 。chu ci zhi wai ,DMRT1can yu xiong xing sheng shi gan xi bao de sun shang xiu fu 、yi zhi gao wan zhong liu 、wei chi xiong xing sheng shi gan xi bao you si fen lie yu jian shu fen lie de ping heng bing ju diao kong xiong xing sheng shi gan xi bao de duo neng xing 。yin ci ,tan jiu DMRT1zai xiong xing sheng shi gan xi bao zhong de gong neng ji zhi ,ke yi wei di gao dong wu wei chuan yo chong 、dong wu ji bing mo xing he wu chong bao hu di gong li lun zhi dao 。zai ben yan jiu zhong wo men shou ci yan jiu le DMRT1dui shan yang xiong xing sheng shi gan xi bao fen hua he zi wo geng xin guo cheng zhong de ying xiang ,yi ji DMRT1zai gao wan sheng shi mian yi zhong fa hui de gong neng 。shi yan jie guo ru xia :1.DMRT1ji yin zai bu ru dong wu zhong an fan cun zai ju bu tong wu chong zhong xu lie gao du bao shou ,duo xu lie bi dui de jie guo fa xian 6chong bu ru dong wu de DMRT1ji yin xu lie yi zhi xing da dao le 86.42%。dui yu ji he xin jie gou yu DMjie gou yu fen xi biao ming ,bu tong wu chong de an ji suan xu lie ji ben yi zhi ,shui ming DMjie gou yu zai bu tong wu chong zhong tong yang ju you gao du bao shou xing 。wei le tan jiu DMRT1zai shan yang bu tong zu zhi de biao da qing kuang ,mian yi ying guang ran se jie guo biao ming DMRT1zai shan yang gao wan zhong te yi biao da 。ran se ji ding liang PCRjin yi bu fa xian DMRT1zai 1yue ling shan yang gao wan de biao da liang hen di ,3yue ling gao wan qi shi jian shu fen lie hou DMRT1biao da ming xian shang diao ,zhi ge ti cheng shou ji biao da shui ping qu yu wen ding 。ji ci ,li yong mian yi ying guang gong ran se jie shi le DMRT1zhu yao ding wei zai wei fen hua he fen hua zhong de xiong xing sheng shi gan xi bao zhong ,dan shi zai qi dong jian shu fen lie de jing mu xi bao zhong que mei you biao da 。gen ju yi shang jie guo ,wo men ren wei DMRT1zhu yao zai xiong xing sheng shi gan xi bao zhong fa hui gong neng ,dui yu mGSCsde fen hua he zeng shi fa hui chong yao zuo yong 。2.FUW-TetO-Dmrt1man bing du zai ti pei ge rtTAfu zhu zhi li zhuai ran xiong xing sheng shi gan xi bao ji yong sheng hua de mGSCs-I-SBxi bao ji ,shai shua chu zui kuo you dao DMRT1biao da de Doxnong du 。EDU、shuang ying guang su mei jian ce 、mian yi ying guang ran se 、Western blotting、ji ding liang PCRjin yi bu jie shi DMRT1dui yu shan yang xiong xing sheng shi gan xi bao fa yo guo cheng de gong neng 。zai 1μg/mL Doxyou dao tiao jian xia ,xiong xing sheng shi gan xi bao zeng shi neng li ji fen hua shui ping xian zhe zeng gao ,jian shu fen lie xiang guan biao ji xiang ying xia diao 。wei jia suan yin qi xiong xing sheng shi gan xi bao de jian shu fen lie ,dan guo biao da DMRT1yi zhi jian shu fen lie de fa sheng tong shi mGSCsde zeng shi biao ji PCNAji fen hua biao ji c-KITbiao da shang diao 。yin ci DMRT1yi fang mian cu jin xiong xing sheng shi gan xi bao de zeng shi he fen hua ,ling yi fang mian yi zhi you RAyin qi de jian shu fen lie yi chang qi shi ,cong er wei chi jing yuan xi bao you si fen lie he jian shu fen lie de ping heng 。3.DMRT1zai gao wan sheng shi mian yi zhong fa hui chong yao gong neng ,DMRT1tong guo yi zhi TLRsxin hao tong lu cong er jiang di xiong xing sheng shi gan xi bao zhong yan zheng fan ying de fa sheng 。shRNAgan rao gao wan xiong xing sheng shi gan xi bao zhong DMRT1de biao da ,zhuai lu zu ce xu ji mian yi zu hua shi yan jie guo xian shi mGSCszhong TLR4ji NF-κBbei ji huo 。ELISAji Western blottingjian ce fa xian xiong xing sheng shi gan xi bao nei cu yan yin zi TNFαji IL-6de biao da shang diao 。mian yi gong chen dian ji shuang ying guang su mei jian ce shi yan jin yi bu jie shi DMRT1qiao mu PLZFdan bai ,er zhe gong tong yi zhi TLR4he NF-κBde zhuai lu bing cu jin xiong xing sheng shi gan xi bao shu liang de hui bu 。yu ci tong shi ,TUNELran se ji liu shi xi bao shu fa xian DMRT1yi zhi you yan zheng fan ying yin qi de xiong xing sheng shi gan xi bao de diao wang 。zeng ge yi shang jie guo biao ming DMRT1zai cu jin xiong xing sheng shi gan xi bao zi wo geng xin ji wei chi gao wan nei huan jing wen tai zhong de chong yao gong neng ,di gong le zhi liao gao wan sheng shi que xian ji bing de xin sai lu 。4.bai xiao an chu li de xiao shu gao wan biao xian chu yan chong de sheng shi xi bao sun shang ,guo biao da DMRT1xian zhe hui fu le xiong xing sheng shi gan xi bao de shu liang bing ju wei chi jing zi fa sheng de zheng chang jin hang 。mian yi ying guang ran se ji ELISAjian ce de jie guo biao ming DMRT1cu jin xiong xing sheng shi gan xi bao zeng shi biao ji PCNA、VASAji zi wo geng xin xiang guan biao ji PLZFji CSF1Rde biao da 。tong shi mGSCszi wo geng xin xiang guan de xi bao yin zi GDNF、CSF1biao da shang diao ,cong er zheng ming DMRT1dui gao wan nei huan jing de chong jian zhi guan chong yao 。ji yu shuang fen zi ying guang hu bu ji shu (BiFC)he mian yi gong chen dian jian ce ,zui zhong que ren DMRT1dan bai yu PLZFdan bai hu zuo zhu yao shi you DMRT1yu PLZFde BTBjie gou yu hu zuo lai shi xian de 。suo yi DMRT1yu PLZFfa hui xie tong zuo yong can yu xiong xing sheng shi gan xi bao zi wo geng xin xia you tong lu de diao jie 。chu ci zhi wai ,DMRT1he PLZFzuo wei yi yan yin zi you xiao de yi zhi qu xi jing guan nei yan zheng de fa sheng ,wei xiong xing sheng shi xi bao sun shang de xiu fu jian li le liang hao de wei huan jing 。zeng shang suo shu ,wo men shou ci tan jiu le DMRT1zai xiong xing sheng shi gan xi bao zhong de biao da mo shi ,tan suo le DMRT1dui shan yang xiong xing sheng shi gan xi bao fen hua he zi wo geng xin guo cheng de ying xiang ,bing jie shi DMRT1zai sheng shi mian yi zhong fa hui de gong neng 。ji yin xiu shi zai ti zhuai ran shan yang xiong xing sheng shi gan xi bao ,ti nei ti wai zeng ge fen xi 、yan zheng shai shua DMRT1ke neng zuo yong de te yi ba ji yin ji ji hu zuo ji zhi ,wei yan jiu shan yang xiong xing sheng shi gan xi bao de zi wo geng xin 、jing zi fa sheng de ji li di gong xin de ke xue yi ju ;wei gao chan you zhi jia chu de wei chuan 、fan yo he bu yo zheng de fang zhi di gong xin fang an 。
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论文作者分别是来自西北农林科技大学的魏于栋，发表于刊物西北农林科技大学2019-07-11论文，是一篇关于雄性生殖干细胞论文,自我更新论文,分化论文,生殖免疫论文,山羊论文,西北农林科技大学2019-07-11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，资料来自西北农林科技大学2019-07-11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：雄性生殖干细胞论文; 自我更新论文; 分化论文; 生殖免疫论文; 山羊论文; 西北农林科技大学2019-07-11论文;