Department of Orthopedic Surgery, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Center for Musculoskeletal Innovative Research and Advancement (C-MiRA), Tokai University Graduate School, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.
Int J Mol Sci. 2021 Apr 29;22(9):4723. doi: 10.3390/ijms22094723.
Previous work showed a link between Tie2 nucleus pulposus progenitor cells (NPPC) and disc degeneration. However, NPPC remain difficult to maintain in culture. Here, we report whole tissue culture (WTC) combined with fibroblast growth factor 2 (FGF2) and chimeric FGF (cFGF) supplementation to support and enhance NPPC and Tie2 expression. We also examined the role of PI3K/Akt and MEK/ERK pathways in FGF2 and cFGF-induced Tie2 expression. Young herniating nucleus pulposus tissue was used. We compared WTC and standard primary cell culture, with or without 10 ng/mL FGF2. PI3K/Akt and MEK/ERK signaling pathways were examined through western blotting. Using WTC and primary cell culture, Tie2 positivity rates were 7.0 ± 2.6% and 1.9 ± 0.3% ( = 0.004), respectively. Addition of FGF2 in WTC increased Tie2 positivity rates to 14.2 ± 5.4% ( = 0.01). FGF2-stimulated expression of Tie2 was reduced 3-fold with the addition of the MEK inhibitor PD98059 ( = 0.01). However, the addition of 1 μM Akt inhibitor, 124015-1MGCN, only reduced small Tie2 expression ( = 0.42). cFGF similarly increased the Tie2 expression, but did not result in significant phosphorylation in both the MEK/ERK and PI3K/Akt pathways. WTC with FGF2 addition significantly increased Tie2 maintenance of human NPPC. Moreover, FGF2 supports Tie2 expression via MEK/ERK and PI3K/Akt signals. These findings offer promising tools and insights for the development of NPPC-based therapeutics.
先前的工作表明，Tie2 核髓核祖细胞 (NPPC) 与椎间盘退变之间存在关联。然而，NPPC 在培养中仍难以维持。在这里，我们报告了全组织培养 (WTC) 结合成纤维细胞生长因子 2 (FGF2) 和嵌合 FGF (cFGF) 补充，以支持和增强 NPPC 和 Tie2 的表达。我们还研究了 PI3K/Akt 和 MEK/ERK 途径在 FGF2 和 cFGF 诱导的 Tie2 表达中的作用。我们使用年轻的疝出核髓核组织进行了研究。我们比较了 WTC 和标准原代细胞培养，以及是否添加 10ng/mL FGF2。通过 Western blot 检测 PI3K/Akt 和 MEK/ERK 信号通路。使用 WTC 和原代细胞培养，Tie2 阳性率分别为 7.0 ± 2.6%和 1.9 ± 0.3%（= 0.004）。在 WTC 中添加 FGF2 可将 Tie2 阳性率提高到 14.2 ± 5.4%（= 0.01）。添加 MEK 抑制剂 PD98059 可使 FGF2 刺激的 Tie2 表达降低 3 倍（= 0.01）。然而，添加 1 μM Akt 抑制剂 124015-1MGCN 仅会降低小部分 Tie2 表达（= 0.42）。cFGF 同样增加了 Tie2 的表达，但在 MEK/ERK 和 PI3K/Akt 途径中均未导致显著磷酸化。添加 FGF2 的 WTC 可显著增加人 NPPC 中 Tie2 的维持。此外，FGF2 通过 MEK/ERK 和 PI3K/Akt 信号支持 Tie2 表达。这些发现为基于 NPPC 的治疗方法的开发提供了有前景的工具和见解。
