Effects of recombinant adenovirus-mediated adiponectin on human umbilical vein endothelial cell injury and the underlying mechanism

doi:10.3969/j.issn.2095-4344.2017.01.021

Abstract

Abstract:

BACKGROUND: Vascular endothelial cells closely adhere to the blood vessel wall so as to play a crucial role in the regulation of vascular homeostasis.
OBJECTIVE: To analyze the inhibitory effects of recombinant adenovirus-mediated adiponectin overexpression on oxidized low density lipoprotein (ox-LDL)-induced atherosclerotic injury in human umbilical vein endothelial cells, nuclear factor κB (NF-κB) p65 nuclear protein and pro-inflammatory factors in the downstream of signaling pathway.
METHODS: There were four groups in this experiment: human umbilical vein endothelial cells were cultured in the conventional medium without ox-LDL induction as control group; human umbilical vein endothelial cells induced by 30 mg/L ox-LDL for 48 hours as ox-LDL induction group; cells firstly treated with 100 µmol/L pyrrolidine dithiocarbamate (PDTC) for 1 hour, and then induced by 30 mg/L ox-LDL for 48 hours as PDTC with ox-LDL induction group; cells transfected by adenovirus bearing a vector encoding for adiponectin and enhanced green fluorescent protein (Ad-APN-eGFP) (multiplicity of infection=100 pfu/cell) for 2 hours, and then induced by 30 mg/L ox-LDL for 48 hours as Ad-APN-eGFP with ox-LDL induction group. Afterwards, all relative indicators were detected in each group.
RESULTS AND CONCLUSION: Endothelial cell injury models were obtained by ox-LDL induction, and the NF-κB signaling pathway was activated with the increased expression of NF-κB p65 nuclear protein. Ad-APN-eGFP could inhibit the NF-κB signaling pathway, which significantly decreased the expression of NF-κB p65 nuclear protein, as well as the gene and protein expressions of VCAM-1, ICAM-1 and MCP-1 in the downstream of signaling pathway, but significantly increased the gene and protein expressions of endothelial nitric oxide synthase. These results show that Ad-APN-eGFP-tranfected human umbilical vein endothelial cells can relieve ox-LDL-induced pro-inflammatory injury and reduced apoptosis via effectively inhibiting the NF-κB activation, which achieves similar outcomes with the PDTC intervention. In other words, Ad-APN-eGFP and PDTC both can reduce inflammatory reactions such as atherosclerotic injury by inhibiting the NF-κB signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Atherosclerosis, Endothelial Cells, Lipoproteins, LDL, Tissue Engineering

CLC Number:

R394.2

Wang Xue-mei, Wei Qin, Duan Ming-jun, Zhang Chun, Yang Yi-ning. Effects of recombinant adenovirus-mediated adiponectin on human umbilical vein endothelial cell injury and the underlying mechanism[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 115-121.

Figures/Tables 2

2.1 重组腺病毒体外转染效率倒置荧光显微镜下观察细胞的绿色荧光蛋白表达，病毒转染12 h后，MOI= 100 pfu/cell组的细胞开始表达eGFP；eGFP的表达强度随着时间延长、转染复数的增加逐渐增强；人脐静脉内皮细胞在转染后48 h达到高峰。至转染后72 h，eGFP在人脐静脉内皮细胞仍处于高表达，见表2，图2。 2.2 内皮细胞体外损伤模型的建立 2.2.1 rAd-APN-eGFP降低人脐静脉内皮细胞上清液中的促炎因子表达，增加抗炎因子表达 ox-LDL 30 mg/L刺激48 h后，MCP-1、VCAM-1和ICAM-1表达急剧增高。单因素方差分析结果显示，与ox-LDL诱导组相比，分别用PDTC干预和rAd-APN-eGFP转染诱导处理后，培养液中的MCP-1、VCAM-1和ICAM-1水平均下降，低于ox-LDL诱导组。而PDTC干预和rAd-APN-eGFP处理组两组间差异没有显著性。说明PDTC干预和rAd-APN-eGFP对由ox-LDL诱导的人脐静脉内皮细胞分泌产生促炎因子都有抑制，且作用相似。ox-LDL诱导人脐静脉内皮细胞48 h后，ox-LDL诱导组的人脐静脉内皮细胞上清液中的内皮型一氧化氮合酶活性低于空白对照组，差异具有显著性；而PDTC干预+ ox-LDL诱导组细胞上清液中的内皮型一氧化氮合酶活性变化，差异无显著性意义(P=0.426)，rAd-APN-eGFP转染+ ox-LDL诱导 48 h后细胞上清液中内皮型一氧化氮合酶活性和对照组相比升高。证明了rAd-APN-eGFP转导能够增加细胞分泌内皮型一氧化氮合酶，促进内皮细胞产生一氧化氮，减轻内皮细胞损伤，加快内皮细胞修复，见表3。 2.2.2 rAd-APN-eGFP对核因子κB p65核蛋白和通路下游促炎因子表达的作用和ox-LDL诱导组相比，rAd-APN-eGFP转染增加了抗炎因子内皮型一氧化氮合酶的基因表达，降低了促炎因子MCP-1、VCAM-1、ICAM-1的基因表达。rAd-APN-eGFP转染细胞和PDTC具有相近的作用，能够抑制ox-LDL诱导的炎症反应，减轻内皮细胞的损伤(见表4，5)。与正常对照组相比，ox-LDL诱导组核因子κB p65核蛋白水平升高，差异有显著性表达；与ox-LDL诱导组相比，核因子κB抑制剂PDTC干预组和rAd-APN-eGFP转染组，核因子κB p65核蛋白水平降低，促炎因子MCP-1、VCAM-1、ICAM-1的表达降低，抗炎因子内皮型一氧化氮合酶的表达增加，差异有显著性表达；PDTC干预组和rAd-APN-eGFP转染组，2组之间差异没有显著性表达。提示rAd-APN-eGFP转染具有和PDTC相似的抑制核因子κB信号通路激活的作用，同时降低促炎因子表达，增加抗炎因子的表达。见表4-7，图3。"

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