巨核细胞/血小板E1A激活基因阻遏子基因敲除小鼠构建及胎肝巨核细胞诱导分化
Construction of megakaryocyte/platelet Creg1 knockout mice and induced differentiation of megakaryocytes from fetal liver
刘晶 1梅竹 1周婷 1刘春影 1刘丹 1闫承慧 1宋海旭1
作者信息
- 1. 北部战区总医院 寒地心血管病全国重点实验室 心血管内科,辽宁 沈阳 110016
- 折叠
摘要
目的 构建巨核细胞/血小板特异性E1A激活基因阻遏子基因(Creg1)敲除小鼠,并诱导小鼠胚胎胎肝巨核细胞的分化,为研究Creg1 在巨核细胞分化和血小板生理功能中的作用提供实验工具及方法.方法 将雌性Creg1flox/flox小鼠与雄性血小板因子4(Pf4)-Cre+小鼠繁育,获得Creg1flox/wt Pf4-Cre+小鼠,进一步交配获得巨核细胞/血小板特异性Creg1 敲除小鼠(Creg1flox/flox Pf4-Cre+,简写为Creg1-/-).采用聚合酶链式反应及琼脂糖凝胶电泳进行基因型鉴定.取Creg1flox/flox和Creg1-/-孕龄约15d小鼠的胎肝,体外培养胎肝细胞 4d,加入血小板生成素,光镜下观察两组巨核细胞形态和大小的区别.结果 本研究成功构建了Creg1-/-小鼠.与Creg1flox/flox小鼠比较,Creg1-/-小鼠巨核细胞中Creg1 mRNA的表达量显著下降,差异有统计学意义(P<0.05).与Creg1flox/flox小鼠比较,Creg1-/-小鼠胎肝巨核细胞诱导分化4d时,光镜下观察发现,巨核细胞的直径明显变小,差异有统计学意义(P<0.05).结论 Creg1-/-小鼠作为研究巨核细胞和血小板相关疾病发生机制的实验工具鼠,可能揭示尚未发现和阐明的重要病理生理学机制,为临床治疗血小板疾病提供理论基础支持.
Abstract
Objective Megakaryocyte/platelet specific Creg1(cellular repressor of E1A-stimulated genes 1)knockout mice were con-structed,and induce megakaryocyte differentiation in mouse fetal liver.Methods Female Creg1flox/flox mice were bred with male plate-let factor4(Pf4)-Cre+mice to obtain Creg1flox/wt PF4-Cre+mice.Further mating yielded megakaryocyte/platelet-specific Creg1 knock-out mice(Creg1flox/flox Pf4-Cre+,Creg1-/-for short).Genotypes were identified by polymerase chain reaction and agarose gel electropho-resis.Fetal liver of Creg1flox/flox and Creg1-/-mice with gestational age of about 15 days was taken,fetal liver cells were cultured in vitro for 4 days,and throbopoietin was added to observe the difference in the morphology and size of megakaryocytes between the two groups under light microscope.Results In this study,Creg1-/-mice were successfully constructed.Compared with Creg1flox/flox mice,the ex-pression of Creg1 mRNA in megakaryocytes of Creg1-/-mice was significantly decreased,with statistical significance(P<0.05).Com-pared with Creg1flox/flox mice,the diameter of megakaryocytes in fetal liver of Creg1-/-mice decreased significantly on the 4th day of in-duction differentiation,and the difference was statistically significant(P<0.05).Conclusion Creg1-/-mice,as experimental tools to study the pathogenesis of megakaryocyte and platelet-related diseases,may reveal important pathophysiological mechanisms that have not yet been discovered and clarified,and provide theoretical support for clinical treatment of platelet diseases.
关键词
E1A激活基因阻遏子基因/巨核细胞/血小板/分化Key words
Cellular repressor of E1A-stimulated genes 1/Megakaryocytes/Blood platelets/Differentiation引用本文复制引用
基金项目
中国博士后面上基金项目(2022M713856)
出版年
2024
NETL
NSTL
万方数据