Age effects on in vitro differentiation and cytokine levels of macaque bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.25.006

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells attract extensive attention because of good biological characteristics and broad prospects, but the cells gradually show the characteristics of the aging with the increase of individual age or incubation time in vitro. Nonhuman primates have similar biological characteristics with human being, and have unique advantage in the animal model and disease treatment research.
OBJECTIVE: To analyze the difference in proliferation and differentiation of bone marrow mesenchymal stem cells from macaques at different ages and to explore the effect of age on bone marrow mesenchymal stem cells and the possible mechanism.
METHODS: Bone marrow samples from male macaques aged < 3 years and over 20 years were collected through bone marrow puncture, and divided into young group and elder group, with three macaques in each group. Then, bone marrow mesenchymal stem cells were isolated and cultured in vitro, and the morphological changes, proliferation and differentiation ability were observed. Age-related beta-galactosidase staining was performed, and protein microarray and ELISA were used to detect cytokine levels.
RESULTS AND CONCLUSION: With age, the proliferation and differentiation of bone marrow mesenchymal stem cells from the elder macaques were reduced significantly, and the number of senescent cells increased significantly; the levels of interleukin-1b, interleukin-4, interleukin-6, tumor necrosis factor α and vascular endothelial growth factor were elevated obviously, the levels of heparin-binding basic fibroblast growth factor and placental growth factor were reduced. These findings indicate that the body's aging lead to the reduction in the proliferation, differentiation and cytokine secretion of bone marrow mesenchymal stem 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, Aging, Macaca, Cytokines, Tissue Engineering

Chen Yu-hao, Zhu Xiang-qing, Guo De-bin, Lin Qing-keng, He Zhi-xu, Pan Xing-hua. Age effects on in vitro differentiation and cytokine levels of macaque bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 3971-3976.

Figures/Tables 2

2.1 骨髓间充质干细胞的培养及鉴定结果采用贴壁培养法分离培养猕猴骨髓间充质干细胞，幼年组于三四天，老年组于5-7 d开始出现细胞贴壁生长，培养基中仍混有其他细胞，随着继续传代培养，贴壁细胞增多且形态均一，呈漩涡状生长(图1)。流式细胞术检测幼年组和老年组猕猴骨髓间充质干细胞的表面抗原，幼年组骨髓间充质干细胞阳性标记物CD90、CD105、CD73的表达率分别为98.4%、97.6%、99.8%，阴性标记物CD45，HLA-DR的标记率为4.19%、0.225%(图2)，符合骨髓间充质干细胞的表型特征，幼年和老年猕猴骨髓间充质干细胞的表面抗原无显著差异。 2.2 骨髓间充质干细胞的增殖能力将幼年和老年猕猴骨髓间充质干细胞接种于96孔板，连续培养7 d，绘制生长曲线，可观察到老年组猕猴骨髓间充质干细胞从第2天开始增殖能力明显低于幼年组(P < 0.05)，见图3。 2.3 骨髓间充质干细胞的诱导分化用第3代骨髓间充质干细胞进行体外诱导分化实验(图4)，成骨诱导后经茜素红染色，出现红染的钙结节，幼年组诱导形成的钙结节较老年组更多而且致密。成软骨诱导后细胞缩聚，形态不规则，经阿利新蓝染色，出现蓝染的软骨细胞合成的蛋白多糖，幼年组诱导形成的软骨细胞聚集更加紧密，老年组较为松散。成脂诱导后经油红O染色，出现红染的脂滴，幼年组形成成片密集的脂滴，老年组分化形成的脂滴较少。 2.4 β-半乳糖苷酶的染色和计数 β-半乳糖苷酶染色阳性细胞的体积增大，核质比减少，胞浆呈蓝绿色，而阴性细胞不着色。幼年组骨髓间充质干细胞的染色阳性率为(1.33± 1.51)%，明显低于老年组(35.33±3.88)%，提示老年组骨髓间充质干细胞出现衰老的形态学改变，见图5。 2.5 细胞因子水平幼年与老年猕猴骨髓上清中的白细胞介素1b、白细胞介素4、白细胞介素6和肿瘤坏死因子α表达水平差异有显著性意义(P < 0.05)，幼年组细胞因子的表达水平高于老年组(表1)。幼年与老年猕猴骨髓上清中血管生成相关因子有3个存在显著差异，其中幼年组肝素结合碱性成纤维细胞生长因子和胎盘生长因子水平低于老年组，而幼年组血管内皮生长因子水平高于老年组(表2)。"

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