Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation, expansion, and cultivation
Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation, expansion, and cultivation
复制标题
DOI：
10.1016/j.spinee.2013.05.025
复制
发表时间：
2013-11-01
期刊：
影响因子：
4.5
通讯作者：
Lin, Feng-Huei
中科院分区：
文献类型：
--
作者：
Yang, Shu-Hua;Hu, Ming-Hsiao;Lin, Feng-Huei
背景：椎间盘（IVDs）是人体最大的无血管结构；因此，这些圆盘内的细胞可能适应低氧条件。虽然在体外培养中已经证明低氧浓度可以促进IVD细胞合成细胞外基质，但在传统组织培养O-2饱和条件下，IVD细胞的分离、扩增和培养仍然是有害的。目的：研究人退行性髓核（NP）细胞在常氧（Nx: 21% O-2）和缺氧（Hx: 3.5% O-2）条件下分离和扩增过程中的表型差异。研究设计：我们研究了人NP细胞的体外分离、扩增和培养。方法：从接受腰椎间盘手术的患者中获得人类NP组织样本。然后分离、扩增髓核细胞，在常氧或缺氧条件下培养。为了确定常氧扩增的作用是否可逆，我们分离了另一组细胞，在常氧条件下扩增，然后在缺氧条件下培养（Nx -> Hx组）。通过细胞增殖、选定基因的RNA表达和免疫组织化学染色来评价不同条件下人NP细胞的表型行为。结果：Nx -> Hx组ⅱ型胶原和聚集蛋白的表达明显高于常氧组，但明显低于缺氧组。常氧组基质金属蛋白酶(MMP)-2和MMP-13的表达明显高于其他各组。缺氧诱导因子（hif）表达水平在常氧组显著升高；然而，在缺氧组中发现了更大程度的HIF-1 α染色，而在常氧组中发现了更多程度的HIF-2 α染色。结论：分离、扩增、缺氧培养的人退行性NP细胞能较好地保持细胞的再生潜能。在常压条件下分离和扩增过程中观察到的受损特性只能通过后来的缺氧培养部分恢复。人NP细胞在缺氧条件下优越的表型行为可能与HIF-1a的高生成和HIF-2 α的低生成有关。在缺氧条件下分离、扩增和培养的细胞在移植时可能表现出更好的再生结果；因此，人类退行性NP细胞的分离和扩增过程应在缺氧环境中进行。(C) 2013爱思唯尔公司版权所有。
BACKGROUND CONTEXT: Intervertebral discs (IVDs) are the largest avascular structures in the body; therefore, cells within these discs might be adapted to low-oxygen conditions. Although it has been demonstrated that a low oxygen concentration could promote synthesis of the extracellular matrix by IVD cells in the in vitro culture, isolation, expansion, and cultivation of IVD cells under classical tissue culture O-2 saturation could still be detrimental.PURPOSE: To investigate the phenotypic differences between human degenerative nucleus pulposus (NP) cells during isolation and expansion under normoxic (Nx: 21% O-2) or hypoxic (Hx: 3.5% O-2) conditions.STUDY DESIGN: We investigated in vitro isolation, expansion, and cultivation of human NP cells.METHODS: Human NP tissue samples were obtained from patients who underwent lumbar disc surgeries. Nucleus pulposus cells were then isolated, expanded, and cultivated under normoxic or hypoxic conditions. To determine whether the effects of normoxic expansion are reversible, another group of cells was isolated and expanded in normoxic conditions and then cultivated under hypoxic conditions (Nx -> Hx group). Cellular proliferation, RNA expression of selected genes, and immunohistochemical staining were performed to evaluate the phenotypic behaviors of human NP cells under different conditions.RESULTS: Expressions of Type II collagen and aggrecan in the Nx -> Hx group were significantly higher than those in the normoxic group but were significantly lower than those in the hypoxic group. The normoxic group showed higher expression of matrix metalloproteinase (MMP)-2 and MMP-13 than did the other groups. Expression levels of hypoxia-inducible factors (HIFs) were significantly higher in the normoxic groups; however, a greater degree of HIF-1 alpha staining was found in the hypoxic group, whereas a greater degree of HIF-2 alpha staining was found in the normoxic group.CONCLUSIONS: Human degenerative NP cells isolated, expanded, and cultivated in hypoxic conditions could better preserve the cells' regenerative potential. Compromised properties that were observed during isolation and expansion under normoxic conditions could only be partially rescued by later hypoxic cultivation. The superior phenotypic behaviors of human NP cells under hypoxia may be related to higher HIF-1a production and lower HIF-2 alpha production. Cells that are isolated, expanded, and cultivated under hypoxic conditions may show better regenerative results when transplanted; therefore, the isolation and expansion processes of human degenerative NP cells should be managed in a hypoxic environment. (C) 2013 Elsevier Inc. All rights reserved.