蛋白激酶D3血管内皮细胞条件性敲除小鼠的构建及表型分析  

Construction and Phenotypic Analysis of Protein Kinase D3 Vascular Endothelial Cell Conditional Knockout Mice

作　　者：张茜 马军虎 海霞 马睿婷 宁晓茜 马小元 张淑雅 ZHANG Qian;MA Junhu;HAI Xia;MA Ruiting;NING Xiaoqian;MA Xiaoyuan;ZHANG Shuya(Department of Biochemistry and Molecular Biology,College of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China;General Hospital of Ningxia Medical University,First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学基础医学院生物化学与分子生物学系,银川750004 [2]宁夏医科大学总医院,宁夏医科大学第一临床医学院,银川750004

出　　处：《宁夏医科大学学报》2024年第9期873-877,885,共6页Journal of Ningxia Medical University

基　　金：宁夏自然科学基金项目(2021AAC03134);国家自然科学基金项目(82270654);中科院西部之光青年项目(XAB2020YW17)。

摘　　要：目的构建Prkd3血管内皮细胞条件性敲除小鼠并分析其表型,探讨血管内皮细胞上Prkd3条件性敲除小鼠的敲除效率及可能的表型。方法利用Cre/LoxP系统构建血管内皮细胞条件性基因敲除小鼠模型,即分别构建Prkd3flox/flox小鼠和Cdh5-Cre工具小鼠,再将两者进行交配,筛选得到Prkd3血管内皮条件性敲除小鼠(Cdh5-Cre;Prkd3flox/flox,简称Prkd3ΔEC)。在血管内皮细胞内特异性敲除Prkd3,通过鼠尾PCR法进行基因型鉴定;分离肝血窦内皮细胞并用Western blot检测敲除效率,通过功能学及组织病理学方法评估内皮细胞中Prkd3缺失对肝脏的影响。结果成功构建Prkd3ΔEC,敲除效率大于90%。表型分析发现,Prkd3ΔEC小鼠表现出自发性肝纤维化,随年龄增长逐渐加重(P<0.05);通过HE染色和天狼星红染色发现肝脏中胶原纤维增多,血管周围的纤维成分明显增多(P<0.05)。表明血管内皮细胞中Prkd3缺失能够诱发自发性肝纤维化。结论成功构建了Prkd3ΔEC,该条件性敲除小鼠的肝脏存在自发性肝纤维化情况。Objective To construct Prkd3 vascular endothelial cell conditional knockout mice and its phenotypes,to explore the knockout efficiency and possible phenotypes of Prkd3 vascular endothelial cell conditional knockout mice.Methods A vascular endothelial cell conditional knockout mice model was constructed using the Cre/LoxP system,which involved constructing Prkd3flox/flox mice and Cdh5-Cre tool mice respectively.Then the two mice were mating and screened to obtain Prkd3 vascular endothelial conditional knockout mice(Cdh5-Cre;Prkd3flox/flox,abbreviated Prkd3ΔEC),achieving specific knockout of Prkd3 within vascular endothelial cells and conducting genotype identification using PCR method;Hepatic sinusoidal endothelial cells were isolated and their knockout efficiency was detected using Western blot.The impact of Prkd3 deficiency in endothelial cells on the liver was evaluated through functional and histopathological aualysis.Results Prkd3ΔEC were successfully constructed,and knockout efficiency was greater than 90%.Phenotypic analysis revealed that Prkd3ΔEC mice exhibited spontaneous liver fibrosis,which gradually worsens with age;Through HE staining and Sirius red staining,it was found that there was a significant increase in collagen fibers in the liver,especially in the fibrous components around blood vessels(P<0.05).It was suggested that the absence of Prkd3 in endothelial cells could induce spontaneous liver fibrosis.Conclusion Prkd3ΔEC were successfully constructed,the liver of the conditionally knockout mice exhibits spontaneous liver fibrosis.

关 键 词：Prkd3 血管内皮细胞条件性敲除小鼠 CRE/LOXP系统 肝纤维化 

分 类 号：R394.3[医药卫生—医学遗传学]