Anti-oxidative activities of supernatants of human fetal placental mesenchymal stem cells cultured in serum-free medium

doi:10.3969/j.issn.2095-4344.2017.05.020

Abstract

Abstract:

BACKGROUND: Current research on mesenchymal stem cells (MSCs) is mostly focused on its immune
regulatory function, while little is reported on the antioxidant capacity of the cells and culture supernatant.
OBJECTIVE: To investigate the anti-oxidative capacity of the supernatant harvested from human fetal placenta MSCs (fPMSCs) under a condition of serum free culture.
METHODS: fPMSCs were cultured with serum free media, and the supernatants of cells at passages 2-6 were collected at 48 hours after culture. Vitamin C was added into the culture medium, as a positive control, and its concentration was 100 μmol/L. The total antioxidant capacity, scavenging capacity of free radicals and antioxidant enzymatic activities of supernatants were measured.
RESULTS AND CONCLUSION: By comparing anti-oxidative activities of vitamin C and naïve culture medium, supernatants collected from fPMSCs cultures exhibited obvious antioxidant capacities at different extents between passages of cell cultures. The total antioxidant capacity of the culture supernatant was comparable to 40-80 μmol/L vitamin C. In addition, all supernatants derived from cells with different passages displayed capacities to scavenge free radicals, including 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), hydroxyl radical (•OH), superoxide anion radical (O2-). Even more, activities of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase, were also detected in supernatants collected from different passages of fPMSCs. Under the serum-free condition, the culture supernatants of fPMSCs have antioxidant capacities at certain extent. However, the antioxidant components and underlying mechanisms need to be further studied.

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: stem cells, Culture supernatant, Anti-oxidative activities

CLC Number:

R394.2

Fu Xue, Zhang Yu-jie, Yan Xiu-rui, Ma Xiao-na, Liu Xiao-ming, Wei Jun. Anti-oxidative activities of supernatants of human fetal placental mesenchymal stem cells cultured in serum-free medium[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 773-779.

Figures/Tables 3

2.2 人胎盘胎儿侧间充质干细胞培养上清的总抗氧化能力上清中的抗氧化物质可以将Fe3+还原成Fe2+，后者可与菲啉类物质形成稳定的络合物，通过比色法测定出其抗氧化能力的高低。此实验中，各代次上清均具有一定的的总抗氧化能力，且各代次之间的总抗氧化能力存在一定趋势，以P3-P4代总抗氧化能力较高。与空白对照组相比，差异均具有统计学差异(P < 0.01或P < 0.05；图2A)。与维生素C的总抗氧化能力的标准曲线对比可见，人胎盘胎儿侧间充质干细胞培养上清的总抗氧化能力相当于40-80 μmol/L维生素C。为了消除不同时间测定以及不同试剂盒的组间差异，实验将各代次上清总抗氧化能力的绝对值与同组100 μmol/L维生素C进行对比，求出比值(图2B)，而所得结果与使用绝对值的统计结果类似，由此可知实验重复性较好，可信度较高。 2.3 人胎盘胎儿侧间充质干细胞培养上清对DPPH自由基清(DPPH•)除能力 DPPH•是一种以氮为中心的自由基，含有一个单电子，在517 nm处有强烈的吸收峰，其乙醇溶液呈深紫色，加入培养上清后，测定清除DPPH•而引起的吸光度的减少可以反映其抗氧化能力的强弱。实验结果显示不同代次的培养上清均具有一定的清除DPPH·的能力，以P3及P6代清除率较高，且各组清除率绝对数值均超过维生素C组。除P2代外，其清除率与空白对照组相比存在显著差异(P < 0.01或 P；图3A)。通过将人胎盘胎儿侧间充质干细胞培养上清对DPPH•的清除率对比求出比值后发现结果类似(图3B)。 < 0.05 2.4 人胎盘胎儿侧间充质干细胞培养上清抑制O2-的活力单位此实验模拟机体中黄嘌呤与黄嘌呤氧化酶反应系统，产生超氧阴离子自由基，加入电子传递物质及gress显色剂，使反应体系呈现紫红色，可用分光光度计测其吸光度。实验的结果显示格代次上清中抑制O2-的能力以P2及P5代抑制作用较强，其绝对值与空白对照组相比均存在显著差异(P < 0.01；图4A)，且消除组间差异后，结果类似(图4B)。 2.5 人胎盘胎儿侧间充质干细胞培养上清抑制羟自由基 (•OH)活力单位羟自由基是一种氧化能力很强的自由基，其性质很活泼，氧化各种有机物和无机物的速率极快，是造成组织脂质过氧化、核酸断裂、蛋白质和多糖分解的主要因素，与人体衰老、肿瘤、辐射孙损伤和细胞吞噬能力有关。因此，清除羟自由基对抗氧化具有重要意义。实验显示各代次上清对O2-的均具有抑制作用，与空白对照组相比均差异存在显著性意义(P < 0.01)，但从绝对值上看，各代次间无差异(图5A，B)。 2.6 人胎盘胎儿侧间充质干细胞培养上清超氧化物歧化酶活力超氧化物歧化酶是人体内清除超氧阴离子自由基的酶，在维持机体自由基平衡上有重要作用。实验通过模拟黄嘌呤及黄嘌呤氧化酶系统产生超氧阴离子自由基，后者氧化羟胺形成亚硝酸盐，在显色剂作用下呈现紫红色，用可见光分光光度计测定其吸光度。当被测样本中含超氧化物歧化酶时，则亚硝酸盐减少，比色管吸光度值降低。实验显示，除P3，P4代外各代次上清超氧化物歧化酶活力与空白对照组相比差异有显著性意义(P < 0.05；图6A)，但消除了组间差异后，P3，P4代上清超氧化物歧化酶活力与空白对照组相比差异有显著性意义(P < 0.05；图6B)。 2.7 人胎盘胎儿侧间充质干细胞培养上清谷胱甘肽过氧化物酶活力谷胱甘肽过氧化物酶是人体内广泛存在的一种重要的过氧化物分解的酶，它特异地催化还原型谷胱甘肽对氢过氧化物的还原反应。一般认为它在细胞内能清除有害的过氧化物代谢产物，阻断脂质过氧化链锁反应，从而起到保护细胞膜结构和功能完整的作用。实验显示除P6代外，各代次上清谷胱甘肽过氧化物酶活力与空白对照组相比存在显著差异(P < 0.01；图7A)，消除了组间差异后结果也类似(图7B)。"

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