中国组织工程研究

中国组织工程研究 ›› 2018, Vol. 22 ›› Issue (8): 1161-1166.doi: 10.3969/j.issn.2095-4344.0814

• 骨组织构建 bone tissue construction • 上一篇    下一篇

苯肾上腺素对氧化应激损伤成骨细胞形态和Nampt的影响

张  菊，韩立赤，汪义菲，闫晓旭   

  1. 大连大学医学院口腔医学系，辽宁省大连市  116622
  • 收稿日期:2017-11-18 出版日期:2018-03-18 发布日期:2018-03-18
  • 通讯作者: 韩立赤，博士，硕士生导师，副主任医师，大连大学医学院口腔医学系正畸科，辽宁省大连市 116622
  • 作者简介:张菊，女，1995年生，山西省朔州市人，汉族，大连大学医学院口腔医学系在读本科生。
  • 基金资助:

    辽宁省科技厅自然科学基金资助课题(201202008)；2016年度大连大学教学改革项目(G1-65)；大连大学本科生创新教育基金项目(2014124)

Phenylephrine effects on the morphology of osteoblasts and expression of nicotinamide phosphoribosyltransferase under oxidative stress

Zhang Ju, Han Li-chi, Wang Yi-fei, Yan Xiao-xu   

  1. Department of Oral Medicine, Medical College, Dalian University, Dalian 116622, Liaoning Province, China
  • Received:2017-11-18 Online:2018-03-18 Published:2018-03-18
  • Contact: Han Li-chi, M.D., Master’s supervisor, Associate chief physician, Department of Oral Medicine, Medical College, Dalian University, Dalian 116622, Liaoning Province, China
  • About author:Zhang Ju, Department of Oral Medicine, Medical College, Dalian University, Dalian 116622, Liaoning Province, China
  • Supported by:

    the Natural Science Foundation of Liaoning Province Science and Technology Department, No. 201202008; the Teaching Innovation Project of Dalian University in 2016, No. G1-65; the Innovation Education Foundation for the Undergraduates of Dalian University, No. 2014124

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摘要：

文章快速阅读：

文题释义：
苯肾上腺素：苯肾上腺素是α1-肾上腺素受体(α1-Adrenergic receptor，α1-AR)特异性激动剂。α1-AR 广泛分布于机体的器官、组织和细胞中，介导多种生理反应。α1-AR能够通过增加成骨细胞转录因子的启动子或增强子结合蛋白的表达从而诱导其增殖，苯肾上腺素对成骨细胞的生长具有重要作用。
氧化应激损伤成骨细胞：H2O2氧化应激不仅影响成骨细胞的活性还对细胞分化产生作用，氧化应激可减少成骨细胞的成熟和细胞内矿化物质的合成，使其在成骨细胞形态学上表现为数量减少和体积皱缩等。

摘要
背景：已有研究显示苯肾上腺素对放射性损伤的唾液腺细胞和上皮细胞等均具有保护作用，但其对过氧化氢造成氧化应激损伤的成骨细胞作用尚不清楚。
目的：研究苯肾上腺素对过氧化氢造成的成骨细胞氧化应激损伤中烟酰胺磷酸核糖转移酶(nicotinamide phosphoribosyltransferase，Nampt)的调控作用。
方法：原代培养成骨细胞，随机分为空白对照组、单纯H2O2处理组、苯肾上腺素组和苯肾上腺素+H2O2组(1×10-5 mol/L的苯肾上腺素预处理半小时后，300 μmol/L过氧化氢作用于成骨细胞)。不同时间点观察细胞，并于24 h时间点收集细胞，提取细胞总RNA和总蛋白，应用半定量RT-PCR和免疫印迹法(Western blotting)检测成骨细胞mRNA调控和蛋白的表达情况。
结果与结论：①镜下观察可见单纯H2O2处理组较空白对照组细胞数量减少、形态皱缩明显；苯肾上腺素+ H2O2组在处理12，24和48 h后细胞数量较单纯H2O2处理组均明显增多；②RT-PCR结果显示24 h时间点，单纯H2O2处理组较空白对照组Nampt mRNA表达减少31.23%(P < 0.05)；苯肾上腺素+H2O2组较单纯H2O2处理组Nampt mRNA表达增加206.20%(P < 0.05)；③Western blotting结果显示24 h时间点，单纯H2O2处理组较空白对照组Nampt 蛋白表达减少67.98%(P < 0.05)；苯肾上腺素+H2O2组较单纯H2O2处理组Nampt 蛋白表达增加152.25%(P < 0.05)；④提示苯肾上腺素可缓解过氧化氢导致的成骨细胞减少和皱缩，使细胞Nampt mRNA和蛋白表达水平上调，Nampt 基因可能是苯肾上腺素对成骨细胞氧化应激损伤作用的相关调控基因。

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程
ORCID: 0000-0003-2534-6246(张菊)

关键词: 苯肾上腺素, Nampt, 过氧化氢(H2O2), 成骨细胞, 组织块培养法, 细胞增殖, MTT, 基因表达

Abstract:

BACKGROUND: Phenylephrine has been proved to exert a protective effect on radiant-induced salivary gland and epithelial cell injuries, but its effect on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblasts are not fully understood.
OBJECTIVE: To explore the effect of phenylephrine on H2O2-induced oxidative stress in osteoblasts, and to explore the mechanism underlying the regulation by the expression level of nicotinamide phosphoribosyltransferase (Nampt).
METHODS: Primary osteoblasts were cultured and randomly divided into four groups: blank control group, H2O2 group, phenylephrine group, and combination group (0.5 hour pretreatment of 1×10-5 mol/L phenylephrine, and then given 300 μmol/L H2O2). The morphology of osteoblasts was observed at different time points. Osteoblasts were collected after 24-hour culture, and total RNA and protein were then extracted to detect the mRNA and protein expression levels of Nampt by RT-PCR and western blot assay, respectively.
RESULTS AND CONCLUSION: Compared with the blank control group, reduced osteoblasts and evident cell shrinks were observed in the H2O2 group, while the number of osteoblasts significantly increased in the combined group compared with the H2O2 group at 12, 24 and 48 hours of culture. RT-PCR results showed that the mRNA level of Nampt in the H2O2 group was reduced by 31.23% of that in the blank control group, while the mRNA level of Nampt in the combination group was dramatically increased by 206.20% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Furthermore, western blot assay findings revealed that the protein level of Nampt in the H2O2 group was reduced by 67.98% of that in the blank control group, while the protein level of Nampt in the combination group was increased by 152.25% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Our results indicate that phenylephrine can alleviate the shrink and atrophy of osteoblasts caused by H2O2, thereby exerting protective effect by up-regulating the mRNA and protein levels of Nampt that may be a regulatory gene.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteoblasts, Cell Proliferation, Cell Differentiation, Oxidative Stress, Transaminases, Tissue Engineering

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