重组脂联素对氧化应激状态下内皮细胞NF-κB、iNOS和NO信号表达的影响

发布时间：2018-06-12 20:07

本文选题：内皮细胞 + 第三丁基过氧化氢　；参考：《福建医科大学》2009年硕士论文

【摘要】： 目的:研究NF-κB-iNOS-NO信号通路在第三丁基过氧化氢(Tert-butylhydroperoxide,t-BHP)诱导内皮细胞损伤中的作用,在此基础上进一步研究重组脂联素对氧化应激状态下内皮细胞的NF-κB-iNOS-NO信号表达的影响。方法:体外培养人脐静脉内皮细胞(HUVEC),用t-BHP诱导刺激,模拟体外内皮细胞氧化应激损伤的细胞模型,通过MTT法观察t-BHP作用后细胞的存活率,Griess化学显色法检测氧化损伤过程中细胞内NO水平,同时应用Western blot技术检测胞浆及胞核核因子κB片段p65(NF-κBp65)蛋白、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)蛋白表达水平的变化。应用iNOS抑制剂左旋硝基精氨酸甲酯(N~G-Nitro-L-arginine Methyl Ester,L-NAME)预处理并检测细胞内NO水平。随后用含脂联素基因的重组腺病毒处理,(选MOI100,作用时间48h)观察重组脂联素对t-BHP诱导内皮细胞损伤的保护作用。结果:MTT显示t-BHP(12.5-100umol/L)作用不同时间(30-120min)后可明显抑制内皮细胞的生长,并呈现一定的时效和量效关系,50umol/L浓度的t-BHP处理60min时胞浆iNOS蛋白达高峰水平,处理30min时胞核NF-κB活化片段p65蛋白达高峰水平,iNOS抑制剂L-NAME(600umol/L)预处理细胞24h后可明显抑制内皮细胞胞内NO水平的升高。加入含脂联素基因的重组腺病毒处理,(选MOI100,作用时间48h)干预处理后可抑制t-BHP诱导的内皮细胞胞内NO水平的升高,并且重组脂联素能抑制t-BHP诱导的内皮细胞胞核NF-κBp65及胞浆iNOS蛋白的表达水平。结论:(1)t-BHP可抑制内皮细胞的生长,在次过程中伴随细胞内NO水平的升高,(2)氧化应激NF-κB-iNOS-NO信号通路可能是t-BHP诱导内皮细胞发生损伤的重要通路之一。(3)重组脂联素可以抑制t-BHP诱导内皮细胞损伤,同时抑制细胞NO水平,(4)重组脂联素可能通过抑制细胞内NF-κB活化,胞浆iNOS表达水平来抑制NO水平,最终减轻氧化应激诱导的内皮细胞损伤。
[Abstract]:Aim: to investigate the role of NF- 魏 B-iNOS-NO signaling pathway in endothelial cell injury induced by Tert-butylhydroperoxidet-BHP (Ding Ji), and to investigate the effect of recombinant adiponectin on the expression of NF- 魏 B-iNOS-NO signal in endothelial cells under oxidative stress. Methods: human umbilical vein endothelial cells (HUVECs) were cultured in vitro and stimulated by t-BHP. MTT assay was used to observe the survival rate of the cells treated with t-BHP and the level of no in the cells during oxidative injury was detected by Griess chemical staining. Meanwhile, the cytoplasm and nuclear factor 魏 B fragment p65NF- 魏 B p65 protein were detected by Western blot. Expression of inducible nitric oxide synthase (iNOS) protein. NG-Nitro-L-arginine methyl ester (NG-Nitro-L-arginine Methyl EsterL-NAME-NAME), an iNOS inhibitor, was used to pretreat and detect the level of no in the cells. Then the protective effect of recombinant adiponectin on t-BHP induced endothelial cell injury was observed with recombinant adenovirus treated with adiponectin gene (MOI 100 for 48 h). Results after treated with t-BHPX 12.5-100 umol / L for 30 to 120 min, the growth of endothelial cells was significantly inhibited, and the expression of iNOS protein reached the peak level in 60min treated with t-BHP at 50 umoll / L concentration in a time-dependent and dose-dependent manner. After treated with 30min, the p65 activation fragment of nuclear NF- 魏 B reached the peak level. Pretreatment with L-NAME-600 umol / L (iNOS inhibitor) for 24 h significantly inhibited the increase of no level in endothelial cells. After treated with recombinant adenovirus containing adiponectin gene (MOI100 for 48h), the increase of no level in endothelial cells induced by t-BHP was inhibited. The recombinant adiponectin inhibited the expression of NF- 魏 B p65 and iNOS protein in the nucleus of endothelial cells induced by t-BHP. Conclusion the growth of endothelial cells could be inhibited by TBP. The NF- 魏 B-iNOS-NO signaling pathway of oxidative stress may be one of the important pathways of t-BHP induced endothelial cell injury. The recombinant adiponectin can inhibit the injury induced by t-BHP. The recombined adiponectin may inhibit the level of no by inhibiting the activation of NF- 魏 B and the expression of iNOS in the cytoplasm, and ultimately alleviate the injury of endothelial cells induced by oxidative stress.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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