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作 者:石嘉琛 黄玲巍 田玲 马芳芳 康碧静 杨迪[1] 乔自林[1,2] 阿依木古丽·阿不都热依木 SHI Jia-chen;HUANG Ling-wei;TIAN Ling;MA Fang-fang;KANG Bi-jing;YANG Di;QIAO Zi-lin;AYIMUGULI Abudureyimu(Animal Cell Technology Innovation Center of Gansu Province,Biomedical Research Center,Northwest Minzu University,Lanzhou 730030,China;Key Laboratory of Biotechnology&Bioengineering of the State Ethnic Affairs Commission,Lanzhou 730030,China;College of Life Science and Engineering,Northwest Minzu University,Lanzhou 730030,China)
机构地区:[1]西北民族大学生物医学研究中心,甘肃省动物细胞技术创新中心,甘肃兰州730030 [2]生物工程与技术国家民委重点实验室,甘肃兰州730030 [3]西北民族大学生命科学与工程学院,甘肃兰州730030
出 处:《生物技术》2022年第1期15-20,40,共7页Biotechnology
基 金:甘肃省青年科技基金项目(21JR1RA209);甘肃省重点研发计划项目(21JR1RA222);甘肃省科技计划项目(18JR2JA004);中央高校基本科研业务费专项资金(31920210002)。
摘 要:[目的]探究不同浓度miR-2779表达水平变化对Bak1蛋白和mRNA表达的影响。[方法]将miR-2779模拟物(mimics)按照浓度分为30 nmol/L、50 nmol/L、75 nmol/L和100 nmol/L组;抑制剂(inhibitor)分为75 nmol/L、100 nmol/L和120 nmol/L组,分别转染MDCK细胞,48 h后提取细胞总RNA,颈环法反转录miRNA,实时荧光定量PCR技术检测MDCK细胞中miR-2779和Bak1 mRNA表达水平,Western Blotting检测Bak1蛋白表达情况。[结果]与对照组相比,miR-2779 mimics转染组miR-2779表达水平均显著上调,75 nmol/L miR-2779 mimics转染组miR-2779显著上调11.39±0.19倍(P<0.001);75 nmol/L和100 nmol/L组Bak1蛋白表达水平显著下调0.54±0.03、0.45±0.04倍;Bak1 mRNA表达水平均显著下调。miR-2779 inhibitor转染后75 nmol/L组miR-2779无显著变化,100 nmol/L和120 nmol/L组显著下调0.621±0.03、0.649±0.03倍;100 nmol/L miR-2779 inhibitor转染组中Bak1蛋白表达水平上调2.42±0.06倍。[结论]Bak1表达水平与miR-2779表达水平呈现负向调控,75 nmol/L miR-2779 mimics与100 nmol/L miR-2779 inhibitor为转染MDCK细胞的最佳浓度。miR-2779可调控MDCK细胞中的Bak1表达水平,可能通过其他凋亡信号通路上游因子调控Bak1表达,参与细胞凋亡过程。[Objective]The influence of miR-2779 different levels on Bak1 protein and mRNA expression was investigated.[Method]miR-2779 mimics were divided into 30 nmol/L,50 nmol/L,75 nmol/L and 100 nmol/L groups according to concentration.Inhibitor were divided into 75 nmol/L,100 nmol/L and 120 nmol/L groups,respectively.Total RNA was extracted from MDCK cells after transfected 48 hours,and miRNA was reversely transcribed by neck ring method.The mRNA expression levels of miR-2779 and Bak1 in MDCK cells were detected by RT-qPCR,and Bak1 protein expression was detected by Western Blotting.[Result]The expression level of miR-2779 was significantly up-regulated in the miR-2779 mimics group compared with the control group,and was significantly up-regulated 11.39±0.19 times in the 75 nmol/L miR-2779 mimics transfection group(P<0.001).Bak1 protein expression was significantly decreased by 0.54±0.03 and 0.45±0.04 fold in 75 nmol/L and 100 nmol/L groups.Bak1 mRNA expression levels were significantly down-regulated.miR-2779 inhibitor was transfected into MDCK cells after 48 hours,miR-2779 in 75 nmol/L group had no significant change,and miR-2779 in 100 nmol/L and 120 nmol/L groups was significantly down-regulated by 0.621±0.03 and 0.649±0.03 fold.Bak1 protein expression was up-regulated by 2.42±0.06 fold with 100 nmol/L with miR-2779 inhibitor transfected.[Conclusion]Bak1 expression level was negatively regulated with miR-2779 expression level,and 75 nmol/L miR-2779 mimics and 100 nmol/L miR-2779 inhibitor were the optimal concentrations for transfection MDCK cells.miR-2779 may regulate Bak1 expression through other upstream factors of apoptotic signaling pathways,and participate in the apoptosis process of MDCK cells through Bak1.
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