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作者文欢（2019）在《敲降PHC1蛋白对小鼠胚胎干细胞定向心肌细胞分化的影响》一文中研究指出：目的:构建小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)定向心肌细胞分化的模型,探索敲降PHC1蛋白对mESCs定向心肌细胞分化的影响。方法:利用shRNA在mESCs中敲降PHC1蛋白,通过实时荧光定量聚合酶链式反应(Quantitative real-time polymerase chain reaction,qRT-PCR)检测PHC1、多能干性(nanog、sox2、oct4)及三胚层分化基因的表达;蛋白印迹法(Western-Blot,WB)检测PHC1蛋白的表达;免疫荧光染色分析PHC1、nanog蛋白的表达;采用悬滴法(Hanging Drop methods),1%二甲基亚砜(Dimethyl sulfoxide,DMSO)对mESCs进行定向心肌细胞诱导分化,在分化过程中的不同时期、不同阶段采用qRT-PCR检测心肌细胞特异性基因NKX2.5、β-MHC、cTnT、MEF2C、gata4等的表达,Western-Blot、免疫荧光染色检测cTnT表达。结果:1:敲降PHC1蛋白对mESCs多能干性的影响。成功构建PHC1 shRNA,并感染至mESCs中,Western-Blot检测结果示实验组(shPHC1组)PHC1蛋白表达较对照组明显下调;qRT-PCR检测结果示:与对照组(control、Empty vector)相比较,实验组(shPHC1组)中PHC1与多能干性基因(nanog、sox2、oct4)均出现明显下调,且以nanog下调最显著,中胚层相关基因Brachyury、flk1、tbx3明显上调;免疫荧光染色检测结果提示:实验组(shPHC1组)中PHC1蛋白敲降的mESCs中nanog蛋白明显下调,且具有下调共定位现象;细胞表型:实验组(shPHC1组)mESCs细胞边缘变的锐利,形态变的不规则,出现明显分化,而对照组无明显分化。2:敲降PHC1蛋白对mESCs定向心肌细胞分化的影响。实验组(shPHC1组)在心肌细胞定向分化过程中,心肌细胞特异性基因NKX2.5、β-MHC、cTnT、MEF2C、gata4表达上调,且出现跳动的拟胚体(Beating Embryonic body,EB)比率、心肌细胞特异蛋白(cTnT)均高于对照组(P<0.05)。结论:1.敲降PHC1蛋白促进了mESCs向中胚层分化。2.敲降PHC1蛋白促进了mESCs定向心肌细胞分化并提高了分化效率。
Abstract
mu de :gou jian xiao shu pei tai gan xi bao (mouse embryonic stem cells,mESCs)ding xiang xin ji xi bao fen hua de mo xing ,tan suo qiao jiang PHC1dan bai dui mESCsding xiang xin ji xi bao fen hua de ying xiang 。fang fa :li yong shRNAzai mESCszhong qiao jiang PHC1dan bai ,tong guo shi shi ying guang ding liang ju ge mei lian shi fan ying (Quantitative real-time polymerase chain reaction,qRT-PCR)jian ce PHC1、duo neng gan xing (nanog、sox2、oct4)ji san pei ceng fen hua ji yin de biao da ;dan bai yin ji fa (Western-Blot,WB)jian ce PHC1dan bai de biao da ;mian yi ying guang ran se fen xi PHC1、nanogdan bai de biao da ;cai yong xuan di fa (Hanging Drop methods),1%er jia ji ya feng (Dimethyl sulfoxide,DMSO)dui mESCsjin hang ding xiang xin ji xi bao you dao fen hua ,zai fen hua guo cheng zhong de bu tong shi ji 、bu tong jie duan cai yong qRT-PCRjian ce xin ji xi bao te yi xing ji yin NKX2.5、β-MHC、cTnT、MEF2C、gata4deng de biao da ,Western-Blot、mian yi ying guang ran se jian ce cTnTbiao da 。jie guo :1:qiao jiang PHC1dan bai dui mESCsduo neng gan xing de ying xiang 。cheng gong gou jian PHC1 shRNA,bing gan ran zhi mESCszhong ,Western-Blotjian ce jie guo shi shi yan zu (shPHC1zu )PHC1dan bai biao da jiao dui zhao zu ming xian xia diao ;qRT-PCRjian ce jie guo shi :yu dui zhao zu (control、Empty vector)xiang bi jiao ,shi yan zu (shPHC1zu )zhong PHC1yu duo neng gan xing ji yin (nanog、sox2、oct4)jun chu xian ming xian xia diao ,ju yi nanogxia diao zui xian zhe ,zhong pei ceng xiang guan ji yin Brachyury、flk1、tbx3ming xian shang diao ;mian yi ying guang ran se jian ce jie guo di shi :shi yan zu (shPHC1zu )zhong PHC1dan bai qiao jiang de mESCszhong nanogdan bai ming xian xia diao ,ju ju you xia diao gong ding wei xian xiang ;xi bao biao xing :shi yan zu (shPHC1zu )mESCsxi bao bian yuan bian de rui li ,xing tai bian de bu gui ze ,chu xian ming xian fen hua ,er dui zhao zu mo ming xian fen hua 。2:qiao jiang PHC1dan bai dui mESCsding xiang xin ji xi bao fen hua de ying xiang 。shi yan zu (shPHC1zu )zai xin ji xi bao ding xiang fen hua guo cheng zhong ,xin ji xi bao te yi xing ji yin NKX2.5、β-MHC、cTnT、MEF2C、gata4biao da shang diao ,ju chu xian tiao dong de ni pei ti (Beating Embryonic body,EB)bi lv 、xin ji xi bao te yi dan bai (cTnT)jun gao yu dui zhao zu (P<0.05)。jie lun :1.qiao jiang PHC1dan bai cu jin le mESCsxiang zhong pei ceng fen hua 。2.qiao jiang PHC1dan bai cu jin le mESCsding xiang xin ji xi bao fen hua bing di gao le fen hua xiao lv 。
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论文作者分别是来自南华大学的文欢，发表于刊物南华大学2019-10-28论文，是一篇关于多梳家族蛋白论文,小鼠胚胎干细胞论文,定向心肌细胞分化论文,南华大学2019-10-28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，资料来自南华大学2019-10-28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。