Long-term Video-micrographic Observation on Proliferation and differentiation of Human Eosinphil Precursors in Vitro.

在目前的研究中，我们使用了一种新的设备来压缩嗜酸性粒细胞前体细胞，使其精细结构变得更加明显，而不会失去其增殖和分化的能力。从脐血中分离出单个核细胞，在含有10%胎牛血清、重组人IL-3和重组人IL-5的改良Dulbecco培养液中，在含5%CO_2的潮湿环境中培养9天。在标本上放了一个小盖子。使用一种新的装置对细胞进行压缩，在盖子滑块上施加一个叉形杆子，施加的压力通过螺丝调节。这些细胞在上述相同的培养液中培养。将尼康倒置显微镜放置在恒温箱中，在5-9天的培养时间内获得延时视频显微摄影。在本观察期间，大多数嗜酸性粒细胞前体细胞分裂2-3次以产生自由移动的成熟嗜酸性粒细胞。世代时间从17小时8分钟到96小时3分钟不等。在更成熟的嗜酸性粒细胞中观察到更长的世代时间的趋势。嗜酸性粒细胞的运动模式随着其分化而改变：最不成熟的具有圆形或椭圆形核的嗜酸性粒细胞前体除了偶尔抛出宽大的面纱外，几乎没有运动；中等成熟的嗜酸性粒细胞前体反复向一个方向伸展和回缩一个相对狭窄的伪足；成熟的嗜酸性粒细胞运动相对较快，呈圆形后端的宽杆状。本研究表明，人的嗜酸性粒细胞前体在适当的压力下可以继续增殖和分化，这使得其细微的结构变化更容易被看到。

In the present study, we used a new device to compress eosinophil precursors allowing their fine structures to become more visible without losing their ability to proliferate and differentiate. Mononuclear cells separated from cord blood were cultured in Iscove's modified Dulbecco medium containing 10% bovine fetal serum, rhIL-3 and rhIL-5, for 9 days in a humidified atmosphere containing 5% CO_2. Non-adhering cells were harvested and placed in a human fibrin clot. A small cover slide was placed over the specimen. The cells were compressed using a new device with a forked rod applied on the cover slide, the pressure applied was adjusted by a screw. The cells were cultured in the same medium as described above. A Nikon phase-contrast inverted microscope placed in a thermostatic chamber was used to obtain time-lapsed video-micrography, over 5-9 days of culture. During this observation period, most of the eosinophil precursors divided 2-3 times to yield freely moving mature eosinophils. Generation time varied from 17 hours 8 minutes to 96 hours 3 minutes. A tendency towards a longer generation time in more mature eosinophils was observed. Pattern of motility in the eosinophils changed along with their differentiation : the most immature eosinophil precursors with a round or oval nucleus showed little motility except for occasionally throwing a wide veil ; intermediately mature eosinophil precursors repeatedly prodded and retracted a reatively narrow pseudopod in one direction ; mature eosinophils showed relatively fast motion taking a shape of a broad rod with a round posterior end.This study showed that human eosinophil precursors could continue to proliferate and differentiate under appropriate compression which allow their fine strucutral changes to be more easily visualizad.