1Department of Orthopedics, Renmin Hospital, School of Basic Medical Sciences, Wuhan University, Wuhan City, China.
2Department of Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan City, China.
J Neurotrauma. 2019 Aug 1;36(15):2337-2347. doi: 10.1089/neu.2018.6133. Epub 2019 Mar 6.
Ependymal cells (EpCs) are a kind of multi-potent stem cells in the central canal of adult spinal cord, which proliferate following spinal cord injury (SCI). Although they can differentiate into functional neurons , EpC progeny differentiate mainly into astrocytes after SCI, and the mechanism remains unclear. The present study aimed to explore whether neuroinflammation induced by classically activated macrophages (M1) or alternatively activated macrophages (M2) had an effect on EpC proliferation and/or differentiation. EpCs were isolated from intact spinal cord of adult mice and co-cultured with M1 or M2, respectively, . EpC proliferation was detected using a Cell Counting Kit-8 (CCK8) assay and Ki67 staining. Expression of Sox2 (SRY-box 2) in EpCs derived from different groups was detected by immunofluorescence and western blotting. Also explored was whether the mitogen activated protein kinase (MAPK) signaling pathway was involved in EpC proliferation. Immunofluorescence staining of βIII-tubulin and MAP2 were performed to assess the differentiation direction of EpCs in different culture conditions. Immunofluorescence and western blotting assays showed much more Sox2-positive EpCs in the group EpCs-M1 than the group EpCs-M2 ( < 0.01). The percentage of EpCs with positive Sox2 staining was decreased after tumor necrosis factor α (TNFα) antibody was added into the medium of EpCs-M1. Correspondingly, fewer Sox2-positive staining cells were observed in the central canal of TNFα-deficient mice with SCI. M1 co-culture promoted EpC proliferation significantly, which could be downregulated by Sox2 gene silencing ( < 0.01). Interestingly, M1 regulated the expression of Sox2 through the MAPK signaling pathway, especially the activation of ERK and p38 kinase. Co-culture in M2 conditioned medium obviously increased the proportion of βIII-tubulin-positive cells ( < 0.01). Small amounts of MAP2-positive neurons could be detected on day 7 in the M2 group and the control group. M1 conditioned medium could promote EpC proliferation in response to SCI through the TNFα-MAPK-Sox2 signaling pathway; M2 conditioned medium favors EpCs differentiating toward neurons.
室管膜细胞（ependymal cells，EpCs）是成年脊髓中央管内的一种多能干细胞，在脊髓损伤（spinal cord injury，SCI）后增殖。尽管它们可以分化为功能性神经元，但 EpC 祖细胞在 SCI 后主要分化为星形胶质细胞，其机制尚不清楚。本研究旨在探讨经典激活的巨噬细胞（M1）或替代激活的巨噬细胞（M2）诱导的神经炎症对 EpC 增殖和/或分化的影响。从成年小鼠的完整脊髓中分离 EpC，分别与 M1 或 M2 共培养。使用细胞计数试剂盒-8（Cell Counting Kit-8，CCK8）检测 EpC 增殖，用 Ki67 染色检测 EpC 增殖。用免疫荧光和蛋白质印迹检测不同组 EpC 中 Sox2（SRY-box 2）的表达。还探讨了丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）信号通路是否参与 EpC 增殖。在不同培养条件下，通过 βIII-微管蛋白和 MAP2 的免疫荧光染色评估 EpC 的分化方向。免疫荧光和蛋白质印迹结果显示，在 EpCs-M1 组中，Sox2 阳性 EpC 明显多于 EpCs-M2 组（ < 0.01）。TNFα 抗体加入 EpCs-M1 培养基后，EpCs 中 Sox2 染色阳性的细胞比例降低。相应地，在 TNFα 缺陷型 SCI 小鼠的中央管中观察到的 Sox2 阳性染色细胞较少。M1 共培养显著促进 EpC 增殖，而 Sox2 基因沉默可下调 EpC 增殖（ < 0.01）。有趣的是，M1 通过 MAPK 信号通路调节 Sox2 的表达，特别是 ERK 和 p38 激酶的激活。在 M2 条件培养基中培养明显增加了 βIII-微管蛋白阳性细胞的比例（ < 0.01）。在 M2 组和对照组中，第 7 天可以检测到少量的 MAP2 阳性神经元。M1 条件培养基可以通过 TNFα-MAPK-Sox2 信号通路促进 SCI 后 EpC 的增殖；M2 条件培养基有利于 EpC 向神经元分化。
