Edaravone protects bone marrow stromal cells from oxidative injury

doi:10.3969/j.issn.2095-4344.2014.32.008

Abstract

Abstract:

BACKGROUND: Edaravone as an antioxidant protective effect on nerve cells injured by hydrogen peroxide has been confirmed, but its protective effect on oxidative damage to bone marrow stromal cells has not been reported in-depth.
OBJECTIVE: To investigate the regulatory effects of edaravone on oxidative injury to bone marrow stromal cells.
METHODS: Bone marrow samples were extracted from the long bone of New Zealand rabbits by the method of washing the pulp cavity, then subjected to the density gradient centrifugation and adherent screening to obtain bone marrow stromal stem cells in vitro. The bone marrow stromal cells at 3 passage were divided into five groups: blank group, treated with low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% double antibody; dexamethasone group, treated with cell culture medium containing 1×10-7 mol/L dexamethasone; 50, 100, 300 mg/L edaravone groups, cultured in cell culture medium containing 1×10-7 mol/L dexamethasone and 50, 100, 300 mg/L edaravone, respectively. After culture, MTT method and flow cytometry were used to detect the proliferative level and cell cycle of cells.
RESULTS AND CONCLUSION: Compared with the control and dexamethasone groups, edaravone significantly enhanced the cell proliferation. Edaravone played a protective role in bone marrow stromal cells. When the concentration was 50 mg/L, edaravone began to play a regulatory role (P < 0.05), and this effect was certainly associated with the concentration of edaravone. When the concentration was up to 100 mg/L, edaravone showed a better protective role
(P < 0.01). However, with increasing concentration, this protective effect was not further increased, but decreased slightly. Results indicated that high-concentration dexamethasone can induce oxidative injury to bone marrow stromal cells, and edaravone can protect the cells against this oxidative damage by antioxidant role.

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

全文链接：

Key words: bone marrow, mesenchymal stem cells, antioxidants, femur head necrosis, hormones, dexamethasone

CLC Number:

R394.2

Zhang Zhen-yu, Gao Ming, Feng Lei, Cao Yun-tao, Chen Lei, Li Ming-chao, Liu Chao . Edaravone protects bone marrow stromal cells from oxidative injury[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5126-5131.

Figures/Tables 1

2.1 显微镜下观察骨髓基质干细胞形态 2.1.1 原代骨髓基质干细胞显微镜下观察培养液中存在大量大小不一的圆形细胞；24 h后少数细胞开始贴壁生长，48 h后见贴壁生长细胞数量增多，换液去除大量不贴壁生长的血细胞，可见圆形细胞中散在有少数多角形或短梭形细胞，有数个类似成纤维细胞的突起；三四天后梭形细胞和多角形细胞显著增多；五六天后形成细胞集落，数量明显增多，呈现长梭形、纺锤形和多角形，细胞轮廓清晰，排列较紧密；7-9 d后，细胞的生长明显加速，逐步形成大小不一、分散的细胞集落，呈放射状向四周扩展，细胞呈漩涡状或平行排列(图1A)。大约2周细胞生长至铺满培养瓶底，这时，就开以进行细胞传代培养。 2.1.2 传代后的骨髓基质干细胞传代的细胞贴壁时间和增殖速度比原代培养要快，大约24 h就开始贴壁生长，培养8-11 d细胞互相接触形成单层。传代细胞不再以集落生长，而是以单细胞生长，细胞的分裂相多见，胞体大，细胞边界不是十分清楚，胞浆内颗粒多，显示细胞功能活跃，处于活跃的基质合成状态(图1B)。 2.1.3 氧化损伤的骨髓基质干细胞加入高浓度地塞米松后，细胞生长停止，细胞胞体变小，并且开始变形，胞浆内颗粒也减少，细胞数量开始逐渐减少，细胞分裂停止，出现细胞死亡现象(图1C)。 2.1.4 加入依达拉奉的骨髓基质干细胞加入质量浓度为50 mg/L的依达拉奉后，骨髓基质干细胞开始缓慢增长，数量有所增加，细胞形态成正常化发展(图2A)；加入100 mg/L的依达拉奉后，骨髓基质干细胞生长速度明显增加，细胞数量明显增多，细胞形态完整，胞体大，胞浆内颗粒多，显示细胞功能又逐渐转为活跃状态(图2B)，证明保护作用有一定的剂量效应；而加入300 mg/L的依达拉奉后，骨髓基质干细胞的增长速度和细胞状态没有得到进一步的提升，相反，细胞的数量还有所减少(图2C)。 2.2 骨髓基质干细胞表面标记物的检测结果流式细胞仪检测第3代骨髓基质干细胞，结果CD34、CD45、CD29、CD90分别为0.79%、0.18%、96.8%、84.2%。CD34，CD45检测为阴性，CD29，CD90检测为阳性(图3)。 2.3 MTT法检测骨髓基质干细胞的增殖活性 MTT检测各组细胞增殖活性见表1。通过比较可知，与空白组比较，地塞米松组的骨髓基质干细胞增殖活性明显下降(P < 0.01)；依达拉奉加入对地塞米松诱导损伤后的骨髓基质干细胞有保护作用，当依达拉奉浓度为50 mg/L时即发挥保护作用(P < 0.05)，并且保护作用与依达拉奉的质量浓度表现出一定的剂量效应。当依达拉奉质量浓度为100 mg/L时，保护作用明显提高(P < 0.01)；但当继续增加依达拉奉的浓度时，保护的作用没有得到进一步的增加。 2.4 流式细胞仪检测各组骨髓基质干细胞的细胞周期在流式细胞术中，通常用S期或G2/M+S期的百分比作为增殖指数，来表示细胞的增殖情况。通过比较可以看出，与空白组相比，地塞米松组的S期和G2/M+S期细胞百分比明显降低(P < 0.01)；增殖指数较空白组也有所降低；而依达拉奉组较地塞米松组的S期和G2/M+S期细胞百分比有所上升(P < 0.05)。这与MTT法检测的结果是相吻合的，差异也具有显著性意义(表2)。"

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