摘要
目的建立一种心肌组织稳定过表达Hotair的新生小鼠模型并进行验证。方法采用改良后的重叠延伸PCR法从小鼠基因组中获取目的基因Hotair,构建过表达Hotair的慢病毒质粒,并采用高分子聚合物转染法进行包装及浓缩纯化,测序结果显示构建的慢病毒质粒Hotair序列正确。将18只P0期(出生24 h内)小鼠随机分为Hotair过表达组、阴性对照组、空白对照组,每组6只。Hotair过表达组于左侧胸腔第五肋间隙注射浓缩后的Hotair慢病毒10μL,阴性对照组和空白对照组分别注射等量空载病毒和生理盐水;各组注射4 d后处死小鼠,取心脏组织。采用实时定量荧光PCR法检测Hotair及受Hotair协同调控的赖氨酸特异性去甲基化酶1(LSD1)mRNA表达,Western blotting法检测LSD1调控的下游蛋白即组蛋白H3二甲基化的赖氨酸4(H3K4me2)表达。结果Hotair过表达组心肌组织Hotair表达高于阴性对照组和空白对照组,H3K4me2蛋白表达低于阴性对照组和空白对照组(P均<0.01)。Hotair过表达组与阴性对照组心肌组织LSD1 mRNA相对比表达量比较差异无统计学意义(P>0.05)。结论成功应用慢病毒载体建立心肌组织稳定过表达Hotair的新生小鼠模型;该模型构建方法简便快捷、Hotair过表达效果明显,为后续研究Hotair参与心脏疾病的相关机制提供了可靠的动物模型。
Objective A neonatal mouse model with stable overexpression of Hotair in the myocardial tissues was established and verified.Methods The target gene Hotair was obtained from mouse genome by improved overlap extension PCR method to construct the lentiviral plasmid overexpressing Hotair,and the lentiviral plasmid Hotair was packaged and purified by high molecular polymer transfection method.The sequencing results showed that the sequence of the constructed lentiviral plasmid was correct.Eighteen P0 mice(within 24 hours after birth)were randomly divided into the Hotair overexpression group(n=6),negative control group(n=6),and blank control group(n=6).In the Hotair overexpression group,10μL concentrated Hotair lentivirus was injected into the fifth intercostal space of the left thoracic cavity,while the mice in the negative control group and the blank control group were injected with the same amount of unloaded virus and the same amount of normal saline,respectively.On the 4th day after virus injection,the mice were killed and the heart tissues were taken.Real-time quantitative PCR was used to detect the expression of Hotair and lysine specific demethylase 1(LSD1)mRNA regulated by Hotair.The expression of histone H3 dimethylated lysine 4(H3K4me2)regulated by LSD1 was detected by Western blotting.Results The expression of Hotair in the myocardial tissues of Hotair overexpression group was higher than that of negative control group and blank control group,while the expression of H3K4me2 protein was lower than that of negative control group and blank control group(all P<0.01).There was no significant difference in the expression of LSD1 mRNA in the myocardial tissues between Hotair overexpression group and negative control group(P>0.05).Conclusion The neonatal mouse model with stable overexpression of Hotair in the myocardial tissues is successfully established by lentivirus vector;this construction method is simple and rapid,and the effect of Hotair overexpression is obvious,which provides a reliable animal model for
作者
费乔曼
邱曼曼
贾子恒
王雯雯
杨冰
张玲
FEI Qiaoman;QIU Manman;JIA Ziheng;WANG Wenwen;YANG Bing;ZHANG Ling(Tianjin Medical University,Tianjin 300070,China)
出处
《山东医药》
CAS
2020年第23期25-29,共5页
Shandong Medical Journal
基金
天津市自然科学基金青年项目(16JCQNJC12100)。
