摘要
目的建立血管内皮细胞连接蛋白43(connexin 43,Cx43)条件性敲除小鼠模型,并对其血管舒张/收缩功能进行检测。方法将8只Cx43^(flox/flox)小鼠与C57BL/6品系野生型(wild type,WT)小鼠交配,子代小鼠继续与C57BL/6小鼠回交9代进行基因背景转换;再将Cx43^(flox/+)小鼠与血管内皮细胞特异性表达Tie2-Cre重组酶小鼠(以下简称Cre小鼠)交配,获得Tie2-Cre/Cx43^(flox/+)小鼠。利用鼠尾PCR试剂盒进行基因型鉴定,Western Blot法和免疫荧光法检测小鼠肠系膜上动脉(superior mesenteric artery,SMA)中Cx43表达。利用离体张力测定技术检测SMA血管舒张/收缩功能,包括去甲肾上腺素诱导的收缩反应性和乙酰胆碱诱导的舒张反应性。结果PCR电泳和血管蛋白表达检测结果证实Tie2-Cre/Cx43^(flox/+)小鼠成功构建,肠系膜上动脉中Cx43蛋白表达与野生型小鼠相比明显降低(P<0.01)。与WT小鼠相比,部分敲除血管内皮细胞Cx43后使乙酰胆碱诱导的内皮依赖的血管舒张反应性显著降低(P<0.01)。结论采用Cx43^(flox/flox)小鼠与血管内皮细胞特异性表达Tie2-Cre重组酶小鼠成功构建血管内皮细胞条件性敲除Cx43杂合子小鼠,该小鼠的肠系膜上动脉中Cx43表达降低,且舒张反应性明显降低,预期可为研究Cx43及其相关结构在血管功能调节中的作用提供动物模型。
Objective To establish an endothelial conditional connexin 43(Cx43)-knockout mouse model and use it to examine vascular relaxation/constriction.Methods Eight Cx43^(flox/flox)mice were crossed with C57BL/6 wild type(WT)mice and the offspring were backcrossed with C57BL/6 mice.This backcross process was repeated nine times,after which the Cx43^(flox/+)mice were crossed with vascular endothelial-cell-specific Tie2-Cre recombinase mice to obtain Tie2-Cre/Cx43^(flox/+)mice.The genotype was confirmed by mouse tail direct polymerase chain reaction(PCR).Expression of Cx43 in the superior mesenteric artery(SMA)was measured by Western Blot and immunofluorescence.SMA rings were used to measure vascular relaxation and the contractile response to acetylcholine(ACh)and norepinephrine.Results Tie2-Cre/Cx43^(flox/+)offspring were obtained and identified by PCR,Western Blot and immunofluorescence.Cx43 expression and ACh-induced endothelium-dependent relaxation reactivity of the SMA were significantly decreased in Tie2-Cre/Cx43^(flox/+)mice compared with WT mice(P<0.01).Conclusions A mouse model with endothelium-specific Cx43-knockout was successfully established by intercrossing Tie2-Cre mice with Cx43^(flox/flox)mice.Cx43 expression in vascular tissues and the relaxation reactivity of SMA were decreased by conditional Cx43 knockout.These mice may thus provide an animal model for studying the link between Cx43 and vascular function.
作者
彭小勇
周远群
张紫森
邓蒙生
雷艳
李涛
刘良明
王建民
康建毅
杨光明
PENG Xiaoyong;ZHOU Yuanqun;ZHANG Zisen;DENG Mengsheng;LEI Yan;LI Tao;LIU Liangming;WANG Jianmin;KANG Jianyi;YANG Guangming(State Key Laboratory of Trauma,Burns and Combined Injury,Department of Shock and Transfusion,Research Institute of Surgery,Daping Hospital,Army Medical University,Chongqing 400042,China;State Key Laboratory of Trauma,Burns and Combined Injury,Department of Weapon Bioeffect Assessment,Research Institute of Surgery,Daping Hospital,Army Medical University,Chongqing 400042)
出处
《中国实验动物学报》
CAS
CSCD
北大核心
2022年第7期880-886,共7页
Acta Laboratorium Animalis Scientia Sinica
基金
国家自然科学基金青年科学基金项目(81801905)
国家自然科学基金面上项目(82072164)
重庆市基础研究与前沿探索项目(cstc2018jcyj AX0555)。
