血管内皮细胞PDHA1基因特异性敲除鼠构建及其表型功能鉴定  

Construction,phenotype and functional identification of vascular endothelial cell-specific PDHA1 knockout mice

作　　者：郝玮 孙岳 杨安宁[2] 刘太阳 宝瑞 刘耀阳 王秋实 王梦 畅思容 李媛媛 刘志宏 Hao Wei;Sun Yue;Yang Anning;Liu Taiyang;Bao Rui;Liu Yaoyang;Wang Qiushi;Wang Meng;Chang Sirong;Li Yuanyuan;Liu Zhihong(School of Public Health and Management,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院,宁夏回族自治区银川市750004 [2]宁夏医科大学基础医学院,宁夏回族自治区银川市750004

出　　处：《中国组织工程研究》2022年第20期3207-3211,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81960018),项目负责人:刘志宏;国家自然科学基金(82060264),项目负责人:孙岳;国家自然科学基金(82160088),项目负责人:杨安宁;2020年度宁夏高等学校科学研究项目(NGY2020031),项目负责人:郝玮。

摘　　要：背景:PDHA1基因是有氧呼吸链中不可或缺的基因,血管内皮细胞的能量代谢与很多疾病有关。构建血管内皮细胞PDHA1基因敲除鼠,有助于研究能量代谢在血管内皮细胞相关疾病中的作用。目的:繁育血管内皮细胞PDHA1基因特异性敲除小鼠并进行表型鉴定。方法:将PDHA1^((iΔEC/iΔEC))小鼠与PDHA1^((iΔEC/-))小鼠进行合笼繁育,获得更多的PDHA1^((iΔEC/iΔEC))小鼠进行后续实验。利用PCR和琼脂糖凝胶电泳进行基因鉴定,然后利用RT-PCR和Western blot检测敲除PDHA1后与能量代谢相关基因的mRNA及蛋白表达,利用VE-Cadherin与PDHA1双重免疫荧光判断PDHA1基因条件性敲除后PDHA1蛋白表达变化。结果与结论:利用琼脂糖凝胶电泳实验成功检测出子代小鼠基因型,包括PDHA1^((iΔEC/iΔEC))15只和PDHA1^((iΔEC/-))2只小鼠;RT-PCR和Western Blot检测发现PDHA1在转录水平和翻译水平都发生了下降,参与糖酵解途径的HK2蛋白及mRNA表达上升,参与有氧磷酸化途径方向IDH蛋白及mRNA表达下降;VE-cadherin和PDHA1双重免疫荧光共染色检测到PDHA1的表达下降。BACKGROUND:PDHA1 gene is an indispensable gene in the aerobic respiratory chain.The energy metabolism of vascular endothelial cells is related to many diseases.Construction of vascular endothelial cell-specific PDHA1 knockout mice helps to study the role of energy metabolism in diseases related to vascular endothelial cells.OBJECTIVE:To breed vascular endothelial cell-specific PDHA1 knockout mice and identify their phenotypes.METHODS:PDHA1^((iΔEC/iΔEC))mice were co-bred with PDHA1^((iΔEC/-))mice,and more PDHA1^((iΔEC/iΔEC))mice were obtained for subsequent experiments.PCR and agarose gel electrophoresis were used for gene identification.RT-PCR and western blot were used to detect the mRNA and protein expression of genes related to energy metabolism after PDHA1 knockout,respectively.The protein expression of PDHA1 after PDHA1 conditional knockout was determined by double immunofluorescence staining of VE-cadherin and PDHA1.2 PDHA1^((iΔEC/-))mice.Results of reverse transcription PCR and western blot assay showed that the transcription and translation levels of PDHA1 were decreased,the protein and mRNA expressions of HK2 in the glycolysis pathway were increased,and the protein and mRNA expression of IDH in the aerobic phosphorylation pathway were decreased.Double immunofluorescence staining of VE-cadherin and PDHA1 showed a decrease in the expression of PDHA1.

关 键 词：PDHA1基因 双重免疫荧光 Cre^(ERT2)基因 表型验证 基因鉴定 能量代谢 血管内皮细胞 肺纤维化 

分 类 号：R459.9[医药卫生—治疗学] R332[医药卫生—临床医学]