大鼠嗅鞘细胞原代培养克隆样生长的人为干预效应

Intervention on the cloning growth of rat olfactory ensheathing cells of primary culture

目的:通过对大鼠嗅鞘细胞早期原代培养的克隆样生长进行人为干预,观察其对细胞的增殖、贴壁及再分布的作用。方法:实验于2005-08/2006-01在云南省肿瘤研究所完成。①将取自成年SD大鼠嗅球的嗅鞘细胞用差速贴壁法进行纯化。②将纯化后的细胞随机分为干预组和对照组,干预组于干预前1d换液,次日倒掉上清液,加入1.25g/L胰酶,加入量以刚浸没瓶底为宜。置于37℃培养箱消化3min,待镜下示胞体回缩变圆时加入DMEM培养基终止消化,并予以尖吸管反复吹打至镜下见大量细胞悬浮无明显克隆样细胞团为止。随后再加入适量培养基置于培养箱中继续培养。对照组不行任何措施。③采用倒置显微镜下观察两组生长的形态学特点,比较干预组干预前后嗅鞘细胞的生长情况及贴壁分布变化。结果:①干预前两组细胞的形态学观察:用差速贴壁法纯化细胞后,发现细胞早期呈克隆样生长,分布不均匀。②干预后两组细胞的形态学观察:干预组细胞经消化吹打干预后均匀贴壁生长,无明显克隆样生长中心,细胞多呈双极或多极样形态,细胞立体感及折光性好,视野清晰,细胞增殖迅速,数天后即可长满瓶底;对照组细胞分布不均,培养至3周仍无法长满瓶底,仍呈克隆样生长状态,周边区域的细胞数量少,增殖缓慢。结论:嗅鞘细胞原代培养的早期需采取人为干预使细胞均匀分布贴壁生长,从而加快细胞的生长速度,缩短实验耗时。

AIM： To investigate the effect of olfactory ensheathing cells （OECs） on the proliferation, adherence and redistribution of cells by interfering the cloning growth of OECs in early primary culture in rats. METHODS： The experiment was conducted in Yunnan Provincial Department of Tumor from August 2005 to January 2006. ① OECs harvested from olfactory bulbs of adult SD rats were purified by different adherence velocity. ② Purified cells were randomly divided into interventional group and control group. Samples in the interventional group were exchanged of the culture fluid at one day before intervention, and the supernatant of which was replaced on the next day with 1.25 g/L pancreatin as just covered the bottom of bottle, which was placed in culture board at 37℃ for 3 minutes. DMEM nutrient medium was added to end the digestion while the cell body recovered and turned to be rounded under the microscope. A pointed pipette was used to boast and aspirate repeatedly until there were no obvious cloning cell groups suspending on most of the cells. Proper nutrient medium was added into the sample, and the culture was continued in the culture board. Samples in the control group was not interfered. ③ Inverted microscope was adopted to observe the morphous characteristics of growth in both groups, and the changes in growth and distribution of adherence of OECs before and after the intervention was compared. RESULTS： ①Observation on the morphous of cells in both groups before intervention： After the cells were purified with different adherence velocity, cells were in cloning growth in the early stage with uneven distribution. ② Observation on the morphous of cells in both groups after intervention： Cells in the intervention group were in even adherence-growth after boasting and digestion, and there was no obvious cloning growth center. Most of the cells were in dipolar or muhipolar morphous with better stereoscopic sensation and refraction, and the field of vision was clear with rapid proliferation of cells. The bottom was covered by cells in a few days. Cells in the control group were in uneven distribution, which still could not cover the bottom of bottle at the 3^rd week, but were in cloning growth with less cells in the periphery and slowly in proliferation. CONCLUSION： The intervention is useful in adherence-growth of OECs of primary culture in early stage to distribute evenly, which can accordingly enhance the growth rate of cells and reduce the experiment session.