pH对体外培养成年大鼠嗅鞘细胞存活的影响

为了研究不同pH培养环境对体外培养大鼠嗅鞘细胞(OECs)存活的影响,本研究采用成年大鼠嗅球培养OECs,培养第8d用S-100免疫组化鉴定纯度后,改用不同pH值(6.8,7.0,7.4,7.8)的DF12培养液继续,在相差显微镜下观察OECs的形态学变化。在不同pH条件下继续培养第3d时采用MTT方法检测不同pH环境中的细胞活力,并用碘化丙啶(PI)和Ho-echst33342荧光双重染色观察细胞死亡率。结果显示:大鼠OECs纯度为(70±4)%;pH7.4组OECs生长旺盛,pH6.8组及pH7.0组OECs生长增殖受到不同程度的抑制,出现死亡细胞;pH7.8组OECs广泛死亡;MTT试验表明,pH7.4组的OD值显著高于其他各pH组。PI/Hoechst33342双重荧光染色显示,在pH6.8、pH7.0和pH7.8组死亡OECs的百分比分别为(27.6±7.8)%、(31.7±7.2)%和(62.3±5.6)%。在pH7.4组未见到死亡的OECs。研究结果表明OECs对酸、碱性环境改变均较敏感,尤其是碱性环境。

INFLUENCE OF pH ON VIABILITY OF OLFACTORY ENSHEATHING CELLS CULTURED IN VITRO

To investigate the influence of pH on the viability of olfactory ensheathing cells (OECs),OECs cells were dissociated from the olfactory bulbs of adult rat. After 8 days of primary culture, the purity of the OECs was evaluated according to the percentage of S-100-immunoreactive cells. The pH of the DF12 medium was adjusted to 6.8, 7.0, 7.4 or 7.8, respectively. The morphological changes of the OECs were examined under a phase contrast microscope. At day 3, MTT assay was performed to assess the viability of the OECs, and propidium iodide (PI) /Hoechst33342 double staining was employed to investigate the death rate of OECs. The results showed that the purity of OECs reached (70 ± 4)%. The OECs in pH 7.4 group showed productive growth condition, whereas the proliferation of the OECs were suppressed in pH 6.8 and pH 7.0 groups with many necrotic OECs. There were large number of necrotic OECs in pH 7.8 group. MTT assay showed that the OD in the pH 7.4 group was significantly higher than that of other pH groups. PI/Hoechst33342 double staining results showed that the percentage of the necrotic OECs in the pH 6.8, pH 7.0 and pH 7.8 groups was (27.6 ± 7.8)% , (31.7 ± 7.2)% and (62.3 ± 5.6)%, respectively. No necrosis was detected in the pH 7.4 group. The present results indicate that OECs are fairly sensitive to the change in pH, especially in the alkaline condition.