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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2017, Vol. 07 ›› Issue (01) : 35 -44. doi: 10.3877/cma.j.issn.2095-1221.2017.01.007

所属专题： 文献；

论著

重组人MG53蛋白对人脐带间充质干细胞氧化损伤的保护作用
黄团结1, 宋及时1, 马珊珊1,†(), 程康1, 邢衢1, 李鹏1, 刘腾飞1, 杨波2, 关方霞3   
  1. 1. 450001　郑州大学生命科学学院
    2. 450052　郑州大学第一附属医院神经外科
    3. 450001　郑州大学生命科学学院；450052　郑州大学第一附属医院神经外科
  • 收稿日期:2016-09-26 出版日期:2017-02-01
  • 通信作者: 马珊珊
  • 基金资助:
    国家自然科学基金(81601078，81471306); 河南省国际人才合作项目(2016GH03，2016GH15)

MG53 protects umbilical cord mesenchymal stem cells from oxidative damage

Tuanjie Huang1, Jishi Song1, Shanshan Ma1,†(), Kang Cheng1, Qu Xing1, Peng Li1, Tengfei Liu1, Bo Yang2, Fangxia Guan3   

  1. 1. Department of Stem Cell Research, School of Life Science, Zhengzhou University, Zhengzhou 450001, China
    2. Stem Cell Research Laboratory, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
    3. Department of Stem Cell Research, School of Life Science, Zhengzhou University, Zhengzhou 450001, China; Stem Cell Research Laboratory, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
  • Received:2016-09-26 Published:2017-02-01
  • Corresponding author: Shanshan Ma
  • About author:
    Corresponding author: Ma Shanshan, Email:
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引用本文：

黄团结, 宋及时, 马珊珊, 程康, 邢衢, 李鹏, 刘腾飞, 杨波, 关方霞. 重组人MG53蛋白对人脐带间充质干细胞氧化损伤的保护作用[J]. 中华细胞与干细胞杂志(电子版), 2017, 07(01): 35-44.

Tuanjie Huang, Jishi Song, Shanshan Ma, Kang Cheng, Qu Xing, Peng Li, Tengfei Liu, Bo Yang, Fangxia Guan. MG53 protects umbilical cord mesenchymal stem cells from oxidative damage[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2017, 07(01): 35-44.

目的

建立人脐带间充质干细胞（hUC-MSCs）的H2O2氧化损伤模型，探讨重组人MG53蛋白（rhMG53）对hUC-MSCs氧化损伤的保护作用。

方法

使用组织块培养法从健康人脐带沃顿胶组织中分离培养hUC-MSCs，流式细胞仪检测细胞表型；CCK-8法检测hUC-MSCs的增殖和H2O2损伤程度。实验分为正常对照组（NC组）、rhMG53组、H2O2组和H2O2+rhMG53组。分别采用CCK-8法、Transwell小室、流式细胞术和β-半乳糖苷酶染色检测rhMG53蛋白对hUC-MSCs增殖、迁移、周期凋亡和衰老的影响。各组、各个时间点的吸光值采用析因设计的方差分析，组间比较采用单因素方差分析。

结果

hUC-MSCs贴壁呈梭型生长，高表达间充质干细胞标志CD44、CD133、HLA-ABC，低表达CD34、CD45；H2O2对hUC-MSCs具有浓度和时间依赖性的损伤作用，确定200μmol/L的H2O2处理16 h为hUC-MSCs氧化损伤的最适条件。与NC组相比，H2O2组细胞增殖（0.994±0.011vs1.331±0.014）、迁移率（8.15﹪±2.47﹪ vs33.34﹪±3.62﹪）和S期细胞比例（29.67﹪±3.69﹪ vs34.33﹪±4.25﹪）显著降低，细胞衰老（44.07﹪±5.26﹪ vs5.7﹪±1.42﹪）和凋亡比例（52.63﹪±5.76﹪ vs4.65﹪±1.23﹪）显著增加，差异具有统计学意义（均P < 0.05）；rhMG53组细胞增殖（1.509±0.086vs1.331±0.014）和迁移率（52.13﹪±5.46﹪ vs33.34﹪±3.62﹪）显著提高，S期比例（35.27﹪±4.79﹪ vs34.33﹪±4.25﹪）变化差异无统计学意义（P> 0.05），细胞衰老（3.92﹪±1.31﹪ vs5.7﹪±1.42﹪）和凋亡比例（3.96﹪±1.06﹪ vs4.65﹪±1.23﹪）显著降低，差异具有统计学意义（均P< 0.05）。与H2O2组相比，H2O2+ rhMG53组细胞增殖（1.252±0.056vs0.994±0.011）、迁移率（18.93﹪±3.12﹪ vs 8.15﹪±2.47﹪）和S期比例（34.84﹪±3.45﹪ vs29.67﹪±3.69﹪）显著提高，细胞衰老（17.89﹪±2.64﹪ vs44.07﹪±5.26﹪）和凋亡比例（4.65﹪±1.23﹪ vs17.63﹪±2.56﹪）显著降低，差异具有统计学意义（均P< 0.05）。

结论

rhMG53对H2O2造成的hUC-MSCs氧化损伤有保护作用。

关键词: 磷蛋白类, 脐带, 间质干细胞, 创伤和损伤, 保护
Objective

To establish a H2O2oxidative damage model of human umbilical cord mesenchymal stem cells (hUC-MSCs) , and to investigate the protective effects of recombinant human MG53 protein (rhMG53) on hUC-MSCs oxidative damage.

Methods

hUC-MSCs were isolated from human umbilical cord tissue collected from healthy donors, and the cell phenotype was determined by flow cytometry; the H2O2-treated P3 hUC-MSCs were evaluated by CCK-8 method. The cells were allocated into four groups: normal control group (NC group) , rhMG53 group, H2O2group and H2O2+ rhMG53 group. The effect of rhMG53 on hUC-MSC proliferation, migration, senescence, cell cycle and apoptosis was respectively detected by CCK-8 assay, Transwell assay, flow cytometry andβ-galactosidase staining. Cell proliferation was analyzed by the variance analysis of factorial design, and the single factor variance analysis was used to compare the differences between groups.

Results

The P3 hUC-MSCs were adherent and spindle shaped, and expressed mesenchymal stem cell markers CD44, CD133, HLA-ABC, with low expression of CD34 and CD45. H2O2inhibitedhUC-MSC growth in a concentration and time-dependent manner.When hUC-MSCs were treated with 200μmol/L H2O2for 16 h, the proliferation (0.994±0.011vs1.331±0.014), migration (8.15﹪±2.47﹪ vs33.34﹪±3.62﹪) and percentage of cells in S phase (29.67﹪±3.69﹪ vs34.3﹪3±4.25﹪) decreased, while the proportion of senescent cells (44.07﹪±5.26﹪ vs5.7﹪±1.42﹪) and apoptosis (52.63﹪±5.76﹪ vs4.65﹪±1.23﹪) significantly increased in the H2O2group compared with the NC group. The proliferation (1.509±0.086vs1.331±0.014) and migration (52.13﹪±5.46﹪ vs33.34﹪±3.62﹪) of cells in the rhMG53 group increased significantly. The proportion of cells in S phase (35.27﹪±4.79﹪ vs34.33﹪±4.25﹪) was similar (P> 0.05) , and the proportion of senescent cells (3.92﹪±1.31﹪ vs5.7﹪±1.42﹪) and apoptosis (3.96﹪±1.06﹪ vs4.65±1.23﹪) was significantly decreased in the rhMG53 group (P< 0.05) . Compared with the H2O2group, thecell proliferation (1.252±0.056vs0.994±0.011) , migration ability (18.93﹪±3.12﹪ vs8.15﹪±2.47﹪) and the proportion of cells in S phase (34.84﹪±3.45﹪ vs29.67﹪±3.69﹪) of H2O2+ rhMG53 group was significantly increased, the proportion of senescent cells (17.89﹪±2.64﹪ vs44.07﹪±5.26﹪) and apoptosis (4.65﹪±1.23﹪ vs17.63﹪±2.56﹪) was significantly decreased (P < 0.05) .

Key words: Phosphoproteins, Umbilical cord, Mesenchymal stem cells, Wounds and injuries, Protection
图1 倒置光学显微镜下观察体外培养的hUC-MSCs形态(×100)
图2 P3代hUC-MSCs免疫表型分析流式图
图3 倒置光学显微镜观察H2O2处理的hUC-MSCs形态变化(×100)
表1 不同浓度H2O2处理对hUC-MSCs增殖的影响（OD450， ± s）
H2O2/（μmol/L） t（培养）/h
0 8 16 24 32 40
0 0.221±0.011 0.481±0.012 0.546±0.013 0.866±0.058 1.216±0.035 1.486±0.063
50 0.224±0.019 0.517±0.031 0.588±0.017 0.831±0.046 1.225±0.116 1.331±0.014
100 0.243±0.018 0.451±0.012 0.561±0.011 0.755±0.051 1.255±0.027 1.342±0.018
200 0.236±0.016 0.311±0.012 0.361±0.019 0.494±0.035 0.856±0.046 1.027±0.050
300 0.234±0.018 0.231±0.013 0.245±0.014 0.326±0.032 0.501±0.062 0.601±0.049
400 0.228±0.014 0.208±0.013 0.217±0.018 0.219±0.013 0.213±0.019 0.215±0.018
F值 1.806 245.197 996.897 127.538 161.689 473.773
P值 0.186 0.000 0.000 0.000 0.000 0.000
表2 CCK-8检测不同处理组的hUC-MSCsA值的变化（OD450， ± s）
分组 t（培养）/h
0 8 16 24 32 40
NC 0.224±0.019 0.369±0.031 0.568±0.036 0.871±0.078 1.259±0.067 1.331±0.014
rhMG53 0.231±0.014 0.415±0.028 0.722±0.022 1.032±0.075 1.476±0.054 1.509±0.086
H2O2 0.236±0.016 0.310±0.012 0.360±0.019 0.527±0.058 0.856±0.046 0.994±0.011
H2O2 + rhMG53 0.221±0.011 0.363±0.041 0.529±0.017 0.799±0.057 1.183±0.053 1.252±0.056
F值 0.787 6.181 121.970 29.063 64.647 51.043
P值 0.534 0.018 0.000 0.000 0.000 0.000
图4 不同处理组迁移细胞数量分析
图5 光学显微镜观察Transwell小室下不同处理组hUC-MSCs的迁移（结晶紫染色，×200）
图6 不同处理组细胞S期百分比分析
图7 流式细胞仪检测不同处理hUC-MSCs的细胞周期变化
图8 不同处理组蓝染细胞数量分析
图9 倒置显微镜观察检测不同处理对hUC-MSCs细胞衰老影响情况（β-半乳糖苷酶染色，×200）
图10 不同处理组凋亡细胞数量分析
图11 流式细胞仪检测不同处理hUC-MSCs的细胞凋亡变化
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