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目的研究嗅成鞘细胞条件培养基(OECCM)对PC12细胞促分化作用及其对-OH自由基损伤分化后细胞的保护作用。方法采用原代分离培养的方法培养和纯化嗅成鞘细胞,收集其培养上清作为OECCM,然后用其培养PC12细胞,培养3 d后,进行细胞形态学观察及-βtubulin免疫细胞化学染色,同时在同一批细胞中加入100μmol/L FeSO4和50μmol/L H2O2作用20 min,再用OECCM继续培养48 h,然后进行MTT对细胞活性进行检测和存活细胞计数。结果用OECCM培养的PC12细胞长有突起,形态酷似神经元,并且-βtubulin免疫细胞化学染色呈阳性,而对照组细胞在同样培养时间里,没有明显的形态变化,-βtubulin染色呈阴性。用FeSO4和H2O2产生的-OH自由基对分化后的PC12细胞进行损伤,发现继续用OECCM培养后,反映细胞活性的A值为0.346 5±0.032,对照组0.201 8±0.034(P<0.01);同时两组细胞存活数目的百分比分别为:实验组为(56.7±5.9)%,对照组为(23.8±7.4)%(P<0.01)。结论嗅成鞘细胞可以分泌有促PC12细胞分化作用的分子和对分化后的细胞在损伤时有保护作用的分子。
Objective To investigate the effect of OECCM on the differentiation of PC12 cells and its protective effects on the differentiation of PC12 cells. Methods Olfactory ensheathing cells were cultured and purified by primary culture. OECCM was harvested from the culture supernatant and then cultured for 3 days. Cell morphology and -βtubulin immunocytochemical staining were performed. The same batch of cells with 100μmol / L FeSO4 and 50μmol / L H2O2 for 20 min, then OECCM continued to culture 48 h, then MTT cell viability was measured and the survival of cell count. Results PC12 cells cultured in OECCM had protuberant protuberances, which were similar to neurons in morphology. Immunocytochemical staining with -βtubulin was positive. In the same culture time, PC12 cells showed no obvious morphological changes, and -βtubulin staining was negative. The differentiated PC12 cells were injured by -OH radical produced by FeSO4 and H2O2. The results showed that A value of cell activity was 0.346 5 ± 0.032 and 0.201 8 ± 0.034 of control group (P <0.01), respectively. The percentage of cell survival in the two groups was (56.7 ± 5.9)% in the experimental group and (23.8 ± 7.4)% in the control group (P <0.01). Conclusion Olfactory ensheathing cells can secrete molecules that promote the differentiation of PC12 cells and protect the differentiated cells from injury.