S100β对炎症条件下嗅鞘细胞的作用及机制

Effect and mechanism of S100β on olfactory ensheathing cells under inflammatory conditions

目的:明确S100β对干扰素-γ(interferon-γ,IFN-γ)诱导的炎症条件下嗅鞘细胞(olfactory ensheathing cells,OECs)的作用,并探讨其发挥作用的机制。方法:将采用改良差速贴壁联合胰酶限时消化3 min法纯化培养的大鼠OECs分为空白对照组、炎症模型组、S100β组等3组,每组种板1×106/ml;空白对照组采用常规培养体系培养OECs,炎症模型组采用重组IFN-γ诱导炎症建立OECs体外炎症模型,S100β组在炎症模型组基础上加入S100β蛋白进行干预;采用免疫荧光染色进行OECs鉴定及纯度分析;利用MTT法检测OECs的增殖情况并筛选IFN-γ诱导OECs炎症模型的适宜浓度;利用TUNEL染色法判断各组OECs的凋亡状况;采用Real-Time PCR检测各组OECs表达炎症相关因子i NOS、IL-1β、TNF-α的mRNA水平。根据以上结果,判断在炎症环境中S100β对OECs的影响及可能机制。结果:采用改良方法原代培养的大鼠OECs能够获得80%纯度;与5 ng/ml的浓度相比,10 ng/ml的IFN-γ对OECs的抑制率更高,凋亡细胞更多;与炎症模型组比较,S100β干预组细胞增殖能力升高,凋亡细胞减少;炎症相关因子i NOS、IL-1β以及TNF-α的基因表达水平下降。结论:S100β可以下调IFN-γ激活的炎症因子i NOS、IL-1β和TNF-α的基因表达水平,提示S100β可能是通过抑制炎症因子的表达从而减少了OECs凋亡的发生。本研究结果为深入研究S100β联合嗅鞘细胞移植对周围神经损伤的修复作用奠定了基础。

Objective： To investigate the effect of S100β on olfactory ensheathing cells（ OECs） under the Interferon-γ（ IFN-γ） induced inflammation,and to explore its related mechanisms. Methods： The modified method was used to purify cultured rat OECs. The experiment was grouped with blank control group,inflammation model group and S100β treated group. The original plant cell dense was 1 × 10^6/ ml. OECs in blank control group were cultivated without any interven-tion,while OECs in inflammation model group were treated with recombinant IFN-γ,and OECs in S100β group were treated with S100β after the inflammation model was set up. Immunofluorescence staining was used for OECs identification and purity analysis. MTT assay was used to test the proliferation ability of OECs and to select the proper concentration of IFN-γ for establishing the OECs inflammation model. TUNEL staining was used to estimate the cell apoptosis in each group. Real-Time PCR was used to detect the gene expression levels of i NOS,IL-1β,and TNF-α. Results： The purified rate of OECs was 80% by the modified primary culture method; Compared with 5 ng / ml concentration of IFN-γ,the 10 ng / ml concentration of IFN-γ showed significantly higher inhibitory rate and more OECs apoptosis; Compared with the inflammation model group,the proliferation ability of OEC in S100β group was increased,and the apoptosis rate was significantly decreased in that group,also the expression level of i NOS,IL-1β,and TNF-α was decreased in that group. Conclusion： S100β can down-regulate the gene expression level of inflammatory factors of i NOS,IL-1β and TNF-α,which suggests that S100β might rescue apoptosis through inhibition of the inflammatory factors＇ expression. These results may provide a proof for the further research about the nerve restoration with olfactory ensheathing cells after the peripheral nerve injury.