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eNOS基因G894T变异对血管内皮细胞产生一氧化氮能力的影响及其意义

Effect of eNOS gene G894T mutation on the ability of vascular endothelial cells to produce nitric oxide and its significance
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摘要 目的探索血管内皮细胞一氧化氮合成酶(eNOS)基因多态性对其产生一氧化氮能力的影响及其与缺血性股骨头坏死(ANFH)发病机制的相关性。方法分别将含894G和894T的eNOS全长CDS片段亚克隆到慢病毒表达载体,转染到血管内皮细胞(HUVEC),在不同时间点检测细胞培养上清中一氧化氮(NO)和鸟苷酸环化酶(cGMP)的含量;用脂质体介导荧光素酶标记的报告质粒pNF-κB-luc与内参对照质粒pRL-TK共转染经LV-eNOS-894G和LV-eNOS-894T及对照慢病毒(LV-NC)转染的HUVEC细胞,待48 h后Western blot法检测各组HUVEC细胞内核因子-κB(NF-κB)、eNOS的蛋白表达。将上述各组HUVEC细胞分别与人成骨细胞hFOB1.19共培养,不同时间点检测细胞培养上清中碱性磷酸酶(ALP)和骨钙素(OCN)的浓度。结果转染eNOS基因(含894G或894T)全长表达慢病毒的细胞培养上清中NO含量和cGMP的含量明显高于空白对照组和空载体组,且894G组NO和cGMP含量显著高于894T组(P<0.01);与空白对照组相比,转染了eNOS基因的细胞中NF-κB/p65和eNOS蛋白表达水平显著增多,且894G组显著高于894T组;上述各组HUVEC细胞分别与hFOB1.19成骨细胞共培养,在同一时间点,转染eNOS基因(含894G或894T)全长表达慢病毒的细胞培养上清中ALP和OCN浓度明显高于空白对照组和空载体组,且894G组的ALP和OCN浓度显著高于894T组。结论 eNOS基因外显子G894T变异通过eNOS-NO通路使血管内皮细胞产生的NO和cGMP下降,影响了NF-κB/p65和eNOS蛋白的表达水平,使骨组织血管供血减少、成骨细胞活性下降,可能是导致股骨头坏死的致病机制之一。 Objective To investigate the relationship between vascular endothelial cell nitric oxide synthase (eNOS) gene polymorphism and the pathogenesis of avascular femoral head necrosis (ANFH). Methods The eNOS full-length CDS fragments, containing 894G or 894T separately, was subcloned into lentiviral expression vector, and then infect the human umbilical vein endothelial cells (HUVEC). The contents of nitric oxide NO and cyclic guanosine monophosphate (cGMP) in cell culture supernatant were detected in a time-dependent manner. The luciferase-labeled reporter plasmid pNF-κB-luc was co-transfected with the reference control plasmid pRL-TK into HUVEC cells infected with LV-eNOS-894G, LV-eNOS-894T, and control lentivirus (LV-NC) for 48 h. The luciferase activity of each group was detected. The expression of nuclear factor (NF)-κB protein and eNOS protein in HUVEC cells were detected by Western blot assay. The HUVEC cells in each group were co-cultured with hFOB1.19 cells, and the concentration of alkaline phosphatase (ALP) or osteocalcin (OCN) in the supernatant were detected at different time points. Results The contents of NO and cGMP in the cell culture supernatant of the full-length lentivirus expressing eNOS gene (containing 894G or 894T) were significantly higher than that in the empty cell group and the empty vector group, and the contents of NO and cGMP in the cell culture supernatant of the 894G group were significantly higher than that of 894T group (P<0.01). Compared with blank cells, the expression levels of NF-κB/p65 protein and eNOS protein were significantly increased in cells expressing eNOS, and the expression of NF-κB/p65 protein in 894G group was significantly increased than 894T group, but there was no difference in eNOS protein expression between the 894G and 894T groups;each group of HUVEC cells were co-cultured with hFOB1.19 osteoblasts, and at each of the same time points, the concentrations of ALP and OCN in the cell culture supernatant expressing lentivirus were significantly higher than that
作者 郑力文 王万春 毛新展 倪江东 魏建伟 陈丁 李丁 毛敏之 Zheng Liwen;Wang Wanchun;Mao Xinzhan;Ni Jiangdong;Wei Jianwei;Chen Ding;Li Ding;Mao Minzhi(Department of Orthopeadics, The Second Xiangya Hospital, Central South University, Changsha 410011, China)
出处 《中国医师杂志》 CAS 2019年第5期677-681,687共6页 Journal of Chinese Physician
基金 湖南省自然科学基金(2016JJ3167).
关键词 内皮 血管 一氧化氮合酶 多态性 单核苷酸 股骨头坏死 Endothelium, vascular Nitric oxide synthase Polymorphism, single nucleotide Femur head necrosis
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