两种不同纯度的嗅鞘细胞在体外存活与生长的比较

Comparison of the cellular survival and growth between olfactory ensheathing cells of two different purities in vitro

比较80%和50%两种纯度的嗅鞘细胞在体外培养条件下的存活与生长,为嗅鞘细胞体内移植选用最佳纯度提供依据。分离、培养并纯化成年SD大鼠嗅鞘细胞,将纯化的嗅鞘细胞和收集到的贴壁成纤维细胞进行约80%和50%的比例混合后接种,继续培养1d或3d。所有细胞于不同时间点行P75免疫细胞化学荧光染色,以鉴定嗅鞘细胞的初始及其后培养过程中数量和纯度的变化。观察细胞状态,计数细胞总数和P75免疫阳性细胞数目,求得各组嗅鞘细胞数量和纯度并行统计学分析。结果显示:两种不同纯度嗅鞘细胞在培养1d时形态正常,3d时纯度为50%的嗅鞘细胞胞体依然饱满,突起更加纤长,数量和纯度亦无明显下降。而纯度为80%的嗅鞘细胞在培养3d时已出现胞体萎缩和突起变短,数量从1d时每一观察区域内的35±13显著下降为23±5(P=0.02),但纯度未发生明显变化。结果表明50%纯度嗅鞘细胞的存活及生长状态优于80%者,提示细胞体内移植时应考虑选取50%纯度的嗅鞘细胞。

In order to compare the survival and growth of olfactory ensheathing cells （OECs） of 80% and 50% purities in vitro, and to present the evidence for the optimal purity of OECs for the transplantation, OECs of adult SD rats were isolated, cultured, purified and mixed for a purity of approximate 80% or 50% with attached fibroblasts. Mixed ceils were cultured for one or three days. All the cultured cells were stained with anti-p75 immunocytochemistry to verify the initial and subsequent numbers and purities of OECs during the cell culture. The morphology of OECs and fibroblasts were observed, the numbers of the total cells and the p75-positive cells were counted, then the purities of OECs were obtained. The mean numbers and purities of the p75-positive cells were analysed statistically. Our results indicated that the OECs with 80% and 50% purities 1 day after culture showed the normal morphology. When the culture time increased to 3 days, OECs with 50% purity had plump cell bodies and long processes, and the mean number and purity of surviving OECs had no dramatical decrease, in comparison with those at 1 day. However, OECs of 80% showed the shrinkage in cell body and the shorter processes, and the mean number of surviving OECs per observing area decreased remarkedly from 35 ± 13 to 23 ± 5 （P = 0.02）, when compared with those at 1 day. We also noted that there was no change in the purity of OECs between 1 and 3 day time points. The present results indicate that the survival and growth of cultured OECs of 50% purity are predominant to those of OECs of 80%, suggesting that 50% OEC suspension should be an optimal candidate for the transplantation into the injured central nervous system.