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

作　　者：刘晶 梅竹 周婷 刘春影 刘丹 闫承慧 宋海旭 LIU Jing;MEI Zhu;ZHOU Ting;LIU Chun-ying;LIU Dan;YAN Cheng-Hui;SONG Hai-xu(State Key Laboratory of Frigid Zone Cardiovascular Disease,Department of Cardiovascular Medicine,General Hospital of Northern Theater Command,Shenyang 110016,China)

机构地区：[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

出　　处：《临床军医杂志》2024年第2期156-158,162,共4页Clinical Journal of Medical Officers

基　　金：中国博士后面上基金项目(2022M713856)。

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Creg1^(flox/flox)小鼠与雄性血小板因子4(Pf4)-Cre^(+)小鼠繁育,获得Creg1flox/wtPf4-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(Creg1^(flox/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Creg1^(flox/flox)和Creg1^(-/-)孕龄约15 d小鼠的胎肝,体外培养胎肝细胞4 d,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Creg1^(flox/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Creg1^(flox/flox)小鼠比较,Creg1^(-/-)小鼠胎肝巨核细胞诱导分化4 d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。Objective Megakaryocyte/platelet specific Creg1(cellular repressor of E1A-stimulated genes 1)knockout mice were constructed,and induce megakaryocyte differentiation in mouse fetal liver.Methods Female Creg1^(flox/flox)mice were bred with male platelet factor 4(Pf4)-Cre^(+)mice to obtain Creg1^(flox/wt)PF4-Cre^(+)mice.Further mating yielded megakaryocyte/platelet-specific Creg1 knockout mice(Creg1^(flox/flox)Pf4-Cre^(+),Creg1^(-/-)for short).Genotypes were identified by polymerase chain reaction and agarose gel electrophoresis.Fetal liver of Creg1^(flox/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 Creg1^(flox/flox)mice,the expression of Creg1 mRNA in megakaryocytes of Creg1^(-/-)mice was significantly decreased,with statistical significance(P<0.05).Compared with Creg1^(flox/flox)mice,the diameter of megakaryocytes in fetal liver of Creg1^(-/-)mice decreased significantly on the 4th day of induction 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 

分 类 号：R392[医药卫生—免疫学] R558[医药卫生—基础医学]