Reduced glutathione effect on the proliferation and neuronal differentiation of bone marrow stromal stem cells

doi:10.3969/j.issn.2095-4344.2016.45.007

Abstract

Abstract:

BACKGROUND: As bone marrow stromal stem cells can be easily obtained and have multi-directional differentiation potential, it is an important approach to obtain neural stem cells for treatment of spinal cord injury through induced differentiation of bone marrow stromal stem cells.
OBJECTIVE: To observe the effects of different concentrations of reduced glutathione on proliferation and neuronal differentiation of bone marrow stromal stem cells.
METHODS: Bone marrow stromal cells were isolated and cultured by limited dilution method. The cloned bone marrow stromal cells were stimulated with reduced glutathione at different concentrations (0, 5, 10, 20, 40 mmol/L) respectively. After 72 hours of culture, proliferation of bone marrow stromal cells was examined by MTT assay. The morphology of bone marrow stromal cells was observed under inverted microscope and the cells were identified by immunofluorescence staining of microtubule associated protein-2 and glial fibrillary acidic protein.
RESULTS AND CONCLUSION: We found that 5 mmol/L reduced glutathione medium could promote the proliferation of bone marrow stromal cells (P < 0.05), but reduced glutathione at 20 and 40 mmol/L inhibited the cell proliferation (P < 0.05). The number and size of cell colonies in the 10 mmol/L reduced glutathione group had no obvious difference from the control group. Reduced glutathione at 10 mmol/L was powerful to induce the neuronal differentiation of bone marrow stromal stem cells, and most cells expressed microtubule associated protein-2 and glial fibrillary acidic protein. A random selection of 5 fields of view was conducted and percentage of bone marrow stromal cells differentiating into neuron-like cells was calculated as (62.0±4.8)%. These results confirmed that low-concentration (5 mmol/L) reduced glutathione can promote the proliferation of bone marrow stromal cells, while 10 mmol/L reduced glutathione has a strong induction of bone marrow stromal cells differentiating into neuron-like cells.

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: Bone Marrow, Mesenchymal Stem Cells, Protein Disulfide Reductase (Glutathione), Cell Differentiation, Neurons, Tissue Engineering

CLC Number:

R394.2

Feng Lin-jie, Gan Hong-quan, Yu Xiang-qian, Yang Jian, Bai Wei-fei, Liu Ying-jie. Reduced glutathione effect on the proliferation and neuronal differentiation of bone marrow stromal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6733-6738.

Figures/Tables 1

2.1 骨髓基质干细胞形态原代细胞接种培养 48 h，可见培养液表面有大量细胞，大部分细胞开始贴壁伸展生长，其胞浆逐渐突起，大多数呈梭形，并可见细胞集落形成，细胞集落大小不一。在初次更换培养液后，细胞迅速增殖，胞体大多呈梭形、不规则形、有多个突起、折光性较强。当细胞生长达到约80%融合时，进行传代接种培养，通过细胞传代和更换培养液，第3代骨髓基质干细胞形态基本较一致，多数呈梭形生长(图1)。 2.2 不同浓度还原型谷胱苷肽对骨髓基质干细胞增殖活性的影响不同浓度还原型谷胱苷肽培养液加入到各组骨髓基质干细胞中，在显微镜下观察，可见低浓度5 mmol/L还原型谷胱苷肽组与对照组相比，细胞集落形成较多，细胞集落较大，细胞多成漩涡状生长；10 mmol/L还原型谷胱苷肽组细胞集落的数量和大小与对照组未见明显差别，随时间推移，细胞胞体缩小，细胞轴突增多；20，40 mmol/L还原型谷胱苷肽组细胞增殖能力下降，细胞集落形成数量未见增多，细胞长度缩小，轴突短小。MTT检测结果经统计分析后显示：5 mmol/L还原型谷胱苷肽组与空白对照0 mmol/L组相比促进细胞增殖(P < 0.05)，10 mmol/L还原型谷胱苷肽组与空白对照0 mmol/L组相比无明显差别(P > 0.05)，但随着还原型谷胱苷肽浓度的增大，逐渐表现出抑制作用，浓度越大抑制作用越明显(P < 0.05)，见图2。 2.3 不同浓度还原型谷胱苷肽对骨髓基质干细胞向神经细胞分化的作用 2.3.1 还原型谷胱苷肽诱导后骨髓基质干细胞的形态学变化骨髓基质干细胞经还原型谷胱苷肽培养液诱导培养24，48，72 h，倒置相差光学显微镜下观察各组细胞形态学变化，发现10 mmol/L 还原型谷胱苷肽组细胞的形态变化更接近神经元细胞，且随着诱导时间的延长，部分骨髓基质干细胞胞体逐渐缩小，细胞逐渐萌生出数量不等的轴突，轴突细长且逐渐向周边延伸，可以与其他细胞相联系，并可见细胞轴突分叉生长，其轴突长度甚至为胞体的数倍(图3)，细胞生存时间较长，而对照组细胞无明显的形态变化。5 mmol/L还原型谷胱苷肽组与对照组相比同样出现细胞增殖能力增加，细胞分化不明显；20 mmol/L还原型谷胱苷肽组和40 mmol/L 还原型谷胱苷肽组细胞与对照组相比出现增殖能力下降，部分细胞出现萎缩。 2.3.2 免疫组织化学荧光双标记染色鉴定不同浓度谷胱甘肽诱导培养72 h行免疫组织化学荧光双标记染色鉴定，结果发现各组差别比较显著，0，5 mmol/L组仅个别细胞表现GFAP和MAP-2阳性，20，40 mmol/L组细胞形态不规则，同样仅有少量细胞表现GFAP和MAP-2阳性；10 mmol/L组可见有较多的细胞表现GFAP和MAP-2阳性(图4)，图4A是在荧光显微镜下采集到的GFAP图像，可见图中位于上方的2个细胞胞体缩小，其中1个细胞有较长的轴突形成，并与下方的细胞有突触联系，位于左上方的细胞则生出了较多的轴突，且彼此之间亦有突触联系，位于下方的骨髓基质干细胞胞体和胞核较大，GFAP呈阴性；图4B中可见标志成熟神经细胞的MAP-2图像，位于上方的2个细胞胞体呈强阳性；图4C是在同一视野所采集到的Hoechst 33342图像；图4D是由A、B、C重叠后的图像，可见位于图片上方的2个细胞胞体呈淡黄色，轴突呈浅红色，表明是由骨髓基质干细胞新生分化而来的神经细胞，位于下方的骨髓基质干细胞未分化，表明部分骨髓基质干细胞在还原型谷胱苷肽作用下可以向神经样细胞方向分化；在显微镜下随机选择5个视野观察并计算骨髓基质干细胞分化为神经样细胞的分化比率为(62.0±4.8)%。"

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