摘要
目的采用改良差速贴壁联合胰酶限时消化法纯化大鼠嗅鞘细胞,并检测嗅鞘细胞的生物活性。方法分离新生7d的SD大鼠嗅球嗅鞘细胞,分组后采用改良的6h和18h两次差速贴壁法联合不同时段（1、3和5min）胰酶消化法纯化培养,观察不同时段纯化的嗅鞘细胞的形态特点及生长情况;应用FITC标记的神经生长因子受体p75抗体（兔抗鼠NGFRp75抗体）检测嗅鞘细胞并评估细胞纯度;应用CCK-8法和酶联免疫吸附法分别检测嗅鞘细胞在纯化后3～15d的增殖活性和分泌脑源性神经营养因子的含量。结果随着细胞的生长,梭形的嗅鞘细胞逐渐增多;改良差速贴壁联合胰酶限时消化1min、3min、5min法所得嗅鞘细胞纯度分别为（57.52±2.13）%、（78.95±2.60）%、（49.01±0.74）%,3min组最明显,组间两两比较差异有统计学意义（P〈0.05）;改良差速贴壁法联合胰酶限时消化1min、3min、5min法所得嗅鞘细胞在纯化后7、9、11d时增殖的细胞数目和脑源性神经营养因子分泌量显示3min组最优,组间比较差异有统计学意义（P〈0.05）。结论改良差速贴壁（6h＋18h）联合胰酶限时消化3min的纯化方法能获得较高纯度的嗅鞘细胞。
Objective To purificate rats,olfactory ensheathing cells ussing modified different adherence combine tryspin digestion method and to monitor the biological activity of olfactory ensheathing cells obtained from the method.Methods Olfactory ensheathing cells were isolated from 7 days old SD rats＇ olfactory bulb and then these cells were pured and cultured by modified different adherence（ 6h ＋ 18h） combine different time（ 1,3,5min） tryspin digestion method.Morphological characteristics and growth condition of olfactory ensheathing cells from different groups were observed.Cell purity was assessed with low-affinity nerve growth factor receptor NGFRp75.Olfactory ensheathing cells＇ proliferation ability after purificated 3 days to 15 days was detected by CCK-8.Brain-derived neurotrophic factor levels from different groups olfactory ensheathing cells after purificated 3 days to 15 days were determined by enzyme-linked immunosorbent.Results After purificated by modified different adherence（ 6h ＋ 18h） combine different time（ 1min,3min,5min） tryspin digestion method,with the cell growing,more and more olfactory ensheathing cells became fusiform in 3 groups and the 3min group was the most obvious.Olfactory ensheathing cells＇ purity at different time（ 1min,3min,5min） were（ 57.52 ± 2.13） %,（ 78.95 ± 2.60） %,（ 49.01 ± 0.74） % and there was statistically significant（ P〈0.05）.Olfactory ensheathing cells obtained after purificated 7 days to 11 days were all in the logarithmic growth phase.The results of cell number and brain-derived neurotrophic factor after purificated 7 days to 11 days comparisons between groups showed that 3min group were the best（ P〈0.05）.Conclusion Modified different adherence（ 6h ＋ 18h） combine tryspin digestion 3min can achieve higher purity olfactory ensheathing cells.Olfactory ensheathing cells obtained after purificated 7 days to 11 days by this method are in the logarithmic phase,in which cell proliferation and brain-derived neurotrophic factor secretion ability are strongest.
出处
《宁夏医科大学学报》
2014年第1期21-24,F0004,共5页
Journal of Ningxia Medical University
基金
银川市科技发展项目(银财发[2011]265号)
宁夏自治区科技支撑项目(宁科计字[2012]17号)
宁夏医科大学博士建设开放课题(KF2010-18)
关键词
嗅鞘细胞
细胞纯度
细胞增殖
脑源性神经营养因子
olfactory ensheathing cells
cell purity
cell proliferation
brain-derived neurotrophic factor
