成年转基因小鼠嗅鞘细胞的培养、纯化及生物学特性

已有多项研究表明,嗅鞘细胞具有修复中枢及外周神经损伤的潜能。我们选用了表达增强型绿色荧光蛋白(enhancedgreenfluorescentprotein,eGFP)的成年小鼠,分离其双侧嗅球嗅神经纤维层及嗅小球层细胞,体外原代培养并予以纯化。同时结合共聚焦、相差显微镜,细胞增殖分析及免疫组织化学鉴定等技术,对其生物学活性进行研究。结果表明:(1)原代培养转基因成年小鼠嗅球嗅鞘细胞(Olfactoryensheathingcells,OECs)15d后,主要存在两种不同形态和免疫组织化学特征的细胞。一种是带有长突起的双极或多极OECs,表达P75~(NIR)(P75lowaffinityneurotrophicreceptor)S100和胶质原纤维酸性蛋白(glialfibrillaryacidicprotein,GFAP)。另一种则是对Thy1.1抗体免疫反应阳性,呈扁平或内皮样形态的成纤维细胞。(2)根据不同类型细胞在未覆层的培养器皿上贴壁速度的差异,我们建立了一种简单易行、不需任何抗体或昂贵仪器的细胞纯化方法,获得了大量高纯度的OECs。(3)在连续纯化培养22d后,OECs仍能保持较高的增殖活性。本实验支持和丰富了OECs发育的相关理论,为进一步体内移植修复CNS损伤提供了理想的材料。

CULTURE, PURIFICATION AND BIOLOGICAL CHARACTERS OF OLFACTORY ENSHEATHING CELLS FROM ADULT TRANSGENIC MICE

Several studies have demonstrated the potential of olfactory ensheathing cells (OECs) for the repair of central and peripheral nerve injury. In this work, ensheathing cells that express the receptor gene coding for enhanced green fluorescent protein (eGFP) from adult mice were iso- lated and purified, and then, its hiological characters were examined in vitro. The work was based on combinations of fluorescence confocal, phase contrast, cell proliferation assay and immunolabel- ing identification. The results showed that (1) two major morphologically and immunohistochemically distinct types of cells were present after 15 days in the primary cultures of adult transgenic mice olfactory nerves and glomerular layers of the olfactory bulb. One cell type was bipolar or multipolar OECs and strained positively for P75 low affinity neurotrophic receptor (P75~(NIR)), S100, and glial fibrillary acidic protein(GFAP). The other type was fibroblasts with flat or endothelial-like shape, and re- acted with antibody against Thy1.1. (2) A simple, inexpensive method for purifying ensheathing cells, in which various harvested cell types showed different rates of attachment to the uncoated culture ware, was developed. This technique neither binds any antibodies nor requires any costly equipment, and yields a large number of highly purified cells. (3) In sequential observations over 22 days in culture, the population of purified cells was maintained and continued to proliferate for longer. This experiment not only supported and advanced the ensheathing cell research but also of- fered ideal materials of in vivo transplantation for the repair of CNS injury.